Thermophilic endo-1,3(4)-beta glucanase (laminarinase) from Rhodothermusmarinus was crystallized by the hanging-drop vapor diffusion method. The needle-like crystals belong to space group P2(1) and contain two protein molecules in the asymmetric unit with a solvent content of 51.75 %. Diffraction data were collected to a resolution of 1.95A and resulted in a dataset with an overall R(merge) of 10.4% and a completeness of 97.8%. Analysis of the structure factors revealed pseudomerohedral twinning of the crystals with a twin fraction of approximately 42%.

Succinate:quinone oxidoreductase (SQR), a di-haem enzyme purified from Rhodothermusmarinus, reveals an HQNO-sensitive succinate:quinone oxidoreductase activity with several menaquinone analogues as electron acceptors that decreases with lowering the redox midpoint potential of the quinones. A turnover with the low-potential 2,3-dimethyl-1,4-naphthoquinone that is the closest analogue of menaquinone, although low, can be detected in liposome-reconstituted SQR. Reduction of the quinone is not stimulated by an imposed K{sup +}-diffusion membrane potential of a physiological sign (positive inside the vesicles). Nor does the imposed membrane potential increase the reduction level of the haems in R. marinus SQR poised with the succinate/fumarate redox couple. The data do not support a widely discussed hypothesis on the electrogenic transmembrane electron transfer from succinate to menaquinone catalysed by di-haem SQRs. The role of the membrane potential in regulation of the SQR activity is discussed.

The subunit II of the caa(3) oxygen reductase from Rhodothermusmarinus contains, in addition to the Cu(A) center, a c-type heme group in the cytochrome c domain (Cyt-D) that is the putative primary electron acceptor of the enzyme. In this work we have combined surface-enhanced resonance Raman (SERR) spectroelectrochemistry, molecular dynamics (MD) simulations and electron pathway calculations to assess the most likely interaction domains and electron entry/exit points of the truncated Cyt-D of subunit II in the reactions with its electron donor, HiPIP and electron acceptor, Cu(A). The results indicate that the transient interaction between Cyt-D and HiPIP relies upon a delicate balance of hydrophobic and polar contacts for establishing an optimized electron transfer pathway that involves the exposed edge of the heme group and guaranties efficient inter-protein electron transfer on the nanosecond time scale. The reorganization energy of ca. 0.7 eV was determined by time-resolved SERR spectroelectrochemistry. The intramolecular electron transfer pathway in integral subunit II from Cyt-D to the Cu(A) redox center most likely involves the iron ligand histidine 20 as an electron exit point in Cyt-D.

The thermohalophilic bacterium Rhodothermusmarinus expresses a caa(3)-type dioxygen reductase as one of its terminal oxidases. The subunit I amino acid sequence shows the presence of all the essential residues of the D- and K-proton channels, defined in most heme-copper oxidases, with the exception of the key glutamate residue located in the middle of the membrane dielectric (E278 in Paracoccus denitrificans). On the basis of homology modeling studies, a tyrosine residue (Y256, R. marinus numbering) has been proposed to act as a functional substitute [Pereira, M. M., Santana, M., Soares, C. M., Mendes, J., Carita, J. N., Fernandes, A. S., Saraste, M., Carrondo, M. A., and Teixeira, M. (1999) Biochim. Biophys. Acta 1413, 1-13]. Here, R. marinus caa(3) oxidase was reconstituted in liposomes and shown to operate as a proton pump, translocating protons from the cytoplasmic side of the bacterial inner membrane to the periplasmatic space with a stoichiometry of 1H(+)/e(-), as in the case in heme-copper oxidases that contain the glutamate residue. Possible mechanisms of proton transfer in the D-channel with the participation of the tyrosine residue are discussed. The observation that the tyrosine residue is conserved in several other members of the heme-copper oxidase superfamily suggests a common alternative mode of action for the D-channel.

The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermusmarinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R. marinus trpB gene. The current work identified an additional selective marker, purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp+ selection was used during the deletion of purA from the R. marinus chromosome. The alternative Ade+ selection was in turn used while deleting the endogenous trpB gene. Since both deletions are unmarked, the purA and trpB markers may be reused. Through the double deletant SB-62 (ΔtrpB ΔpurA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in R. marinus strain SB-62 (ΔtrpB ΔpurA) was demonstrated by targeting putative carotenoid biosynthesis genes, crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5′ and 3′ homologous sequences, respectively. The resulting Trp+ transformants were colorless rather than orange-red. The correct replacement of an internal crtBI fragment with the trpB marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the R. marinus chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination. PMID:21705543

Ten genes encoding novel cellulases with putative signal peptides at the N-terminus, termed pre-LC-CelA–J, were isolated from a fosmid library of a leaf–branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre-LC-CelG have a 14–29 residue long flexible linker (FL) between the signal peptide and the catalytic domain. LC-CelA without a signal peptide (residues 20–261), which shows 76% amino acid sequence identity to Cel12A from Rhodothermusmarinus (RmCel12A), was overproduced in Escherichiacoli, purified and characterized. LC-CelA exhibited its highest activity across a broad pH range (pH 5–9) and at 90 °C, indicating that LC-CelA is a highly thermostable cellulase, like RmCel12A. The crystal structure of LC-CelA was determined at 1.85 Å resolution and is nearly identical to that of RmCel12A determined in a form without the FL. Both proteins contain two disulfide bonds. LC-CelA has a 16-residue FL (residues 20–35), most of which is not visible in the electron density map, probably due to structural disorder. However, Glu34 and Pro35 form hydrogen bonds with the central region of the protein. ΔFL-LC-CelA (residues 36–261) and E34A-LC-CelA with a single Glu34 → Ala mutation were therefore constructed and characterized. ΔFL-LC-CelA and E34A-LC-CelA had lower melting temperatures (Tm) than LC-CelA by 14.7 and 12.0 °C respectively. The Tm of LC-CelA was also decreased by 28.0 °C in the presence of dithiothreitol. These results suggest that Glu34-mediated hydrogen bonds and the two disulfide bonds contribute to the stabilization of LC-CelA. PMID:25426413

The genus Eubacterium currently includes a heterogeneous group of gram-positive, non-spore-forming anaerobic bacilli, many of which are slow growing, fastidious and generally unreactive in biochemical tests. As a consequence, cultivation and identification of isolates are difficult and the taxonomy of the group remains indifferent. In this study, 105 isolates from odontogenic infections, infections associated with dental implants or saliva from healthy subjects and provisionally assigned to the genus Eubacterium were subjected to phenotypic and genotypic analysis. Ninety-one of the isolates were identified as belonging to one of 14 previously described species: Atopobium parvulum (5 isolates), A. rimae (29), Bulleidia extructa (2), Cryptobacterium curtum (1), Dialister pneumosintes (1), Eubacterium saburreum (2), E. sulci (8), E. yurii subsp. yurii (1), Filifactor alocis (3), Lactobacillus uli (1), Mogibacterium timidum (13), M. vescum (6), Pseudoramibacter alactolyticus (6) and Slackia exigua (13). The remaining 14 isolates did not correspond to existing species. This study confirms the diversity of organisms provisionally assigned to the genus Eubacterium by conventional identification methods. This group of organisms is frequently isolated from oral infections but their role in the aetiology of these conditions has yet to be determined.

Eubacterium callanderi is an environmental anaerobic rod-shaped bacterium first isolated in 1998 from an industrial anaerobic digester. We report on the first clinical isolate of E. callanderi, which was recovered from the blood of a patient with a bladder carcinoma. Identification of the organism was made by cell fatty acid chromatographic analysis and 16S rRNA gene sequencing.

The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, Rhodothermus obamensis, was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The ppc gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107,848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of R. obamensis PEPC was closely related to that of PEPC from another thermophile, a Thermus sp., and from a mesophile, Corynebacterium glutamicum, exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the ppc gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in Escherichia coli using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.

Eubacterium spp. and Streptococcus spp. are virulent, commonly identified microorganisms in endodontic infections. The purpose of this study was to use molecular methods to identify these organisms in 22 infected root canals that include eight cases with preoperative clinical symptoms and five cases with a history of diabetes mellitus. The presence of Streptococcus spp. and Eubacterium spp. was examined using two sets of PCR primers specific with multiple species within the respective genera. Positive specimens had their PCR products sequenced and phylogenetically analyzed to identify the specific species. Sixteen specimens (73%) contained Eubacterium spp. and nine (41%) were positive for Streptococcus spp. Eubacterium infirmum was the most prevalent Eubacterium sp. This organism was significantly associated with a history of diabetes (OR = 9.6; P = 0.04). Streptococcus anginosus was the most common Streptococcus sp., but neither it nor any of the other streptococci were significantly associated with the clinical parameters evaluated.

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.

The morphology of Vibrio marinus MP-1 was studied by phase and electron microscopy. The ultrastructure of the vibrio form of V. marinus was found to be typically gram-negative with a trilaminar plasma membrane and cell wall. The coccoid or round bodies noted in otherwise pure cultures of V. marinus were frequently found in early and late stationary phase of growth. The round bodies in ultrathin section were found to contain at least one, and often three or four, cell units. Three types of round bodies were observed in ultrathin section, each differing in size and behavior: “spherules,” “spheres” or the “round body,” and “giant cells” or “macrospheres.” The round bodies appeared to be associated with, or to result from, the constrictive cell division of V. marinus. Images PMID:4895849

Larvicides are used to control sea lamprey (Petromyzon marinus) in the Great Lakes. These larvicides are useful because they are more toxic to sea lamprey than fish species found in the same habitat. The lampricides come from two classes of chemical compounds: (1) halonitrophenols, and (2) halonitrosalicylanilides. Selectivity of the larvicides appears to be based on the differences in the ability of sea lamprey larvae and fishes to detoxify and/or excrete the chemicals. Glucuronide conjugation is an important mechanism for detoxification of these larvicides by fish, and selectivity of larvicides may be due to differences in glucuronyl transferase activity between lamprey and fishes. If more detailed information were available on uptake, metabolism, excretion, and the biochemistry and physiology of lamprey as compared to fishes, it might be possible to design chemicals that would be more selective than those now in use.

Staphylothermus marinus Fiala and Stetter 1986 belongs to the order Desulfurococcales within the archaeal phylum Crenarchaeota. S. marinus is a hyperthermophilic, sulfur-dependent, anaerobic heterotroph. Strain F1 was isolated from geothermally heated sediments at Vulcano, Italy, but S. marinus has also been isolated from a hydrothermal vent on the East Pacific Rise. We report the complete genome of S. marinus strain F1, the type strain of the species. This is the fifth reported complete genome sequence from the order Desulfurococcales.

The chemical, P,P-bis(1-aziridinyl)-N-methylphosphinothioic amide (bisazir), was found in laboratory studies to be an effective sterilant for both sexes of adult sea lampreys (Petromyzon marinus) when given intraperitoneally at a dosage of 100 mg per kilogram of body weight. A total of 300 normal spawning-run sea lampreys and 300 injected with bisazir were released into the Big Garlic River, Marquette County, Michigan, (a small stream divided into five sections by natural barriers), to determine the effect of bisazir on the nesting and spawning behavior of the adults and on the production of larvae. The lampreys constructed and spawned in 95 nests. Sterile adults showed no abnormal nest building or spawning behavior. Sterile males competed effectively with normal males for females. Egg samples taken from nests indicated that eggs in nests where sterile males spawned with sterile or normal females did not hatch, although some embryonic development occurred. Extensive surveys with electric shockers produced no larvae in stream sections where sterile males spawned, but yielded numerous larvae in sections where normal males spawned with normal females. These findings suggest that the release of sterile males may be an effective tool in an integrated approach to control of sea lampreys in the Great Lakes.

The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii) two chiral intermediates were discovered, and (iii) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by the ndhL/ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one [2Fe-2S](2+/1+) and two [4Fe-4S](2+/1+) clusters. Enamidase, a bifunctional Fe-Zn enzyme belonging to the amidohydrolase family, mediates hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to ammonia and (S)-2-formylglutarate. NADH-dependent reduction of the latter to (S)-2-(hydroxymethyl)glutarate is catalyzed by a member of the 3-hydroxyisobutyrate/phosphogluconate dehydrogenase family. A [4Fe-4S]-containing serine dehydratase-like enzyme is predicted to form 2-methyleneglutarate. After the action of the coenzyme B(12)-dependent 2-methyleneglutarate mutase and 3-methylitaconate isomerase, an aconitase and isocitrate lyase family pair of enzymes, (2R,3S)-dimethylmalate dehydratase and lyase, completes the pathway. Genes corresponding to the first three enzymes of the E. barkeri nicotinate catabolism were identified in nine Proteobacteria.

Eubacterium rectale is one of the most abundant bacterial species recovered from human faeces. E. rectale (Hauduroy et al. 1937) appears in the 'List of Bacterial Names with Standing in Nomenclature', but it is noted that the originally proposed type strain, VPI 0989(T), has been lost and its possible replacement by another strain (VPI 0990) from the same faecal sample has never been formally proposed. It is therefore proposed that strain A1-86 (=DSM 17629=NCIMB 14373), isolated from human adult faeces, be formally recognized as the neotype strain of Eubacterium rectale.

Eubacterium cellulosolvens cleaved the flavone C-glucosides homoorientin and isovitexin to their aglycones luteolin and apigenin, respectively. The corresponding isomers, orientin and vitexin, or other polyphenolic C-glucosides were not deglycosylated. E. cellulosolvens also cleaved several O-coupled glucosides of flavones and isoflavones to their corresponding aglycones.

Eubacterium limosum KIST612 is an anaerobic acetogenic bacterium that uses CO as the sole carbon/energy source and produces acetate, butyrate, and ethanol. To evaluate its potential as a syngas microbial catalyst, we have sequenced the complete 4.3-Mb genome of E. limosum KIST612.

Observations on the gland products, gonads, and general characteristics of sexually mature sea lampreys, Petromyzon marinus (Linnaeus), from Lake Huron, and a need to obtain some information on very young larval lampreys, prompted an experiment on the stripping and hatching of eggs. Seventeen specimens were selected from a group of spawning migrants which had been trapped in the Ocqueoc River, Michigan, during June and held in live-cars in the lake until early August.

Background Perkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica, has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States. It is classified as a member of the Perkinsozoa, a recently established phylum considered close to the ancestor of ciliates, dinoflagellates, and apicomplexans, and a key taxon for understanding unique adaptations (e.g. parasitism) within the Alveolata. Despite intense parasite pressure, no disease-resistant oysters have been identified and no effective therapies have been developed to date. Results To gain insight into the biological basis of the parasite's virulence and pathogenesis mechanisms, and to identify genes encoding potential targets for intervention, we generated >31,000 5' expressed sequence tags (ESTs) derived from four trophozoite libraries generated from two P. marinus strains. Trimming and clustering of the sequence tags yielded 7,863 unique sequences, some of which carry a spliced leader. Similarity searches revealed that 55% of these had hits in protein sequence databases, of which 1,729 had their best hit with proteins from the chromalveolates (E-value ≤ 1e-5). Some sequences are similar to those proven to be targets for effective intervention in other protozoan parasites, and include not only proteases, antioxidant enzymes, and heat shock proteins, but also those associated with relict plastids, such as acetyl-CoA carboxylase and methyl erythrithol phosphate pathway components, and those involved in glycan assembly, protein folding/secretion, and parasite-host interactions. Conclusions Our transcriptome analysis of P. marinus, the first for any member of the Perkinsozoa, contributes new insight into its biology and taxonomic position. It provides a very informative, albeit preliminary, glimpse into the expression of genes encoding functionally relevant proteins as potential targets for chemotherapy, and evidence for the presence of a relict

A gene (astA) encoding arylsulfate sulfotransferase (ASST), which transfers a sulfate group from phenolic sulfate esters to phenolic acceptors, was cloned from a Eubacterium A-44 genomic library. The probe (1.5 kb fragment) for the astA gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of ASST, which had been purified from Eubacterium A-44. The astA gene was cloned into the pKF3 vector. Its sequence revealed a 1863 bp open reading frame (ORF) encoding a protein containing 620 amino acids with a secretary signal peptide, and showed 91% homology (identity) to Eubacterium rectale IIIH previously reported. The cloned astA gene was expressed under the T7 promoter of the expression vectors, pET-39b(+) and pET-26b(+), in Escherichia coli BL21 (DE3), and the expressed ASSTs were purified using His Bind column chromatography. The specific activities of the purified ASSTs were 25.6 micromol/min/mg and 37.1 micromol/min/mg, respectively.

The oyster protozoan parasite, Perkinsus marinus, is one of the two important parasites causing severe mortality in the eastern oysters (Crassostrea virginica) on the US east coast. Our recent study suggests that P. marinus cells and its extracellular products (ECP) could scaveng...

Three strains of Eubacterium aerofacien, JCM 10188T, JCM 7790 and JCM 7791, and 178 freshly isolated strains of the Eubacterium aerofaciens group from human faeces were characterized by biochemical tests, cell wall peptidoglycan type and 16S rRNA analysis. The Eubacterium aerofaciens group was divided into four groups by fermentation patterns of sucrose and cellobiose, and were further divided into 16 sub-groups by fermentation patterns of aesculin, salicin and amygdalin. All of the strains of the Eubacterium aerofaciens group were shown to be phylogenetically distantly related to Eubacterium limosum, which is the type species of genus Eubacterium. Eubacterium aerofaciens was shown to have a specific phylogenetic association with Coriobacterium glomerans. All the strains belonging to Eubacterium aerofaciens resembled Coriobacterium glomerans in possessing a high G + C content (60 mol%). Cell wall analysis, however, revealed the presence of different A4 beta (L-Ala)-D-Glu-L-Orn-L-Asp peptidoglycan types. Based on a 16S rRNA sequence divergence of greater than 9% with Coriobacterium glomerans and the presence of a unique peptidoglycan type, a new genus, Collinsella, is proposed for Eubacterium aerofaciens, with one species, Collinsella aerofaciens. The type strain of Collinsella aerofaciens is JCM 10188T.

16S rRNA gene sequences were determined for Eubacterium exiguum and Peptostreptococcus heliotrinreducens. These species were found to be closely related and, together with Eubacterium lentum, to constitute a branch of the Coriobacteriaceae. Two new genera are proposed on the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons: Slackia to include the bile-sensitive species Eubacterium exiguum and P. heliotrinreducens, and Eggerthella to include the bile-resistant Eubacterium lentum. It is proposed that Eubacterium exiguum and Peptostreptococcus heliotrinreducens are transferred to the genus Slackia gen. nov. as Slackia exigua gen. nov., comb. nov. (type strain ATCC 700122T) and Slackia heliotrinireducens gen. nov., comb. nov. (type strain NTCC 11029T), respectively, and Eubacterium lentum is transferred to the genus Eggerthella gen. nov. as Eggerthella lenta gen. nov., comb. nov. with Eggerthella lenta as the type species.

The ability ofAcetobacterium woodii andEubacterium limosum to degrade methyl esters of acetate, propionate, butyrate, and isobutyrate was examined under growing and resting-cell conditions. Both bacteria hydrolyzed the esters to the corresponding carboxylates and methanol under either condition. Methanol was further oxidized to formate under growing but not resting conditions. Unlike the metabolism of phenylmethylethers, no H2 requirement was evident for ester biotransformation. The hydrolysis of methyl carboxylates is thermodynamically favorable under standard conditions and the mixotrophic metabolism of ester/CO2 allowed for bacterial growth. These results suggest that the degradation of methyl carboxylates may be a heretofore unrecognized nutritional option for acetogenic bacteria.

Three 7-wk-old Bobwhite quail were submitted for necropsy to the Douglas branch of the Georgia Poultry Laboratory Network. Grossly, one bird had multiple white foci in the liver and a mild airsacculitis. In this quail there were multiple hepatic granulomas that contained mats of filamentous bacteria easily seen in hematoxylin- and eosin-stained histologic sections. These bacteria were negative with period acid-Schiff and were not acid fast. Bacteria were gram-positive but were most evident on Warthin-Starry silver-stained sections. The appearance and histochemical characteristics of these bacteria are most consistent with Eubacterium tortuosum.

Pseudodiaptomus marinus is a marine calanoid copepod originating of the Indo-Pacific region, who has successfully colonized new areas and it was recently observed in the European side of the Mediterranean Sea as well as in the North Sea. Actually, many questions were posed about the invasive capacity of this copepod in several non-native ecosystems. In this context, the main aim of this study was to investigate the tolerance and the bioaccumulation of metallic stress in the invasive copepod P. marinus successfully maintained in mass culture at laboratory conditions since 2 years. In order to study the metallic tolerance levels of P. marinus, an emergent trace metal, the nickel, was chosen. First, lethal concentrations determination experiments were done for 24, 48, 72 and 96 h in order to calculated LC50% but also to select a relevant ecological value for the suite of experiments. Then, three types of experiments, using a single concentration of nickel (correspond the 1/3 of 96 h-LC50%) was carried in order to study the toxico-kinetics of nickel in P. marinus. Concerning lethal concentrations, we observed that P. marinus was in the same range of sensitivity compared to other calanoid copepods exposed to nickel in the same standardized experimental conditions. Results showed that the uptake of nickel in P. marinus depends from the pathways of entrance (water of food), but also that Isochrysis galbana, used as a food source, has an important bioaccumulation capacity and a rapid uptake of nickel.

We described the first case, to the best of our knowledge, of cutaneous abscess due to Eubacterium lentum in a parenteral drug user, after complete fracture of the right femor. The case underlines the importance of carefully performed microbiological tests, due to the peculiar cultural needs of the micro-organism.

Sea lampreys (Petromyzon marinus L., 1758) are nocturnal, so experiments evaluating their behaviour to chemosensory cues have typically been conducted at night. However, given the brief timeframe each year that adult P. marinus are available for experimentation, we investigated whether P. marinus exposed to a 12 h shifted diurnal cycle (reversed photoperiod) could be tested in a darkened arena during the day and show the same response to chemosensory cues as natural photoperiod P. marinus that were tested during the night. Ten replicates of 10 P. marinus, from each photoperiod, were exposed to deionized water (negative control), 2-phenylethylamine hydrochloride (PEA HCl, putative predator cue), or P. marinus whole-body extract (conspecific alarm cue). All P. marinus demonstrated a significant avoidance response to both cues. No significant differences were found in avoidance to PEA HCl between photoperiods. Avoidance of P. marinus whole-body extract was significantly stronger in natural compared with reversed photoperiod P. marinus. The use of reversed photoperiod subjects is suitable for examining the presence or absence of avoidance in response to novel chemosensory alarm cues, or the change in the magnitude of antipredator response. Studies investigating the natural magnitude of antipredator response should use natural photoperiod experimental subjects.

Echinogammarus marinus constitutes the most abundant amphipod species in Fucus spp. assemblages from many North Atlantic estuaries. However, there are some doubts about the real use of fucoids by the amphipod. Whilst some studies report the ingestion of Fucus vesiculosus by E. marinus, others suggest that the amphipod preference for fucoids is mostly related to sheltering rather than feeding, due to the high phlorotannin content of brown algae. The purpose of the present work was to disentangle this issue by checking the consumption rate and feeding preference of E. marinus on F. vesiculosus, its preferential habitat, and on Ulva intestinalis, a green algae abundant in the Mondego estuary (Western Coast of Portugal) and usually considered as highly palatable for herbivores. In a 2-stage laboratorial setup, fresh disks of the two types of algae were offered to E. marinus for three days. Consumption rates were estimated from differences between algal and animal initial and final fresh weights using a control correction factor, while preference was tested by differences in algal consumption rates when no choice was offered (stage 1) and when the two algae were offered simultaneously (stage 2). Results showed that E. marinus effectively consumed fresh F. vesiculosus in much higher amounts than U. intestinalis and significantly preferred to consume F. vesiculosus over U. intestinalis. Therefore, feeding habits must be one of the factors related to the close association of the amphipod with F. vesiculosus, although other factors may also be involved (e.g. sheltering).

The distribution and abundance of ferric iron in larval lampreys (Petromyzon marinus L.) were investigated using light microscopy and the Prussian blue stain. Animals from various watersheds contained different concentrations of iron, although the sites of deposition were the same for all animals. A major portion of iron is within adipose tissue, while the liver, and cartilage contain predominantly low to trace amounts of iron, respectively. Iron is associated with fibrous connective tissue in several places in the body, and this association may have particular significance in the inner ear. Iron is also located in cells of the meninges. The presence of iron in the epithelial cells of the posterior intestine may reflect elimination of the metal through the extrusion of iron-loaded cells into the intestinal lumen. Iron within mucous cells of the epidermis, suggest elimination of iron during mucous secretion. Iron-loaded cells of bipolar shape are also present in the epidermis, but are particularly prominent around the branchiopore. Low concentrations of iron are observed within in melanin-containing macrophages (melano-macrophages) in regions of iron absorption, erythrophagocytosis, and haemopoiesis. High levels of iron in the epithelia and lumina of pronephric tubules are concomitant with degeneration of this organ. These data are evidence of the wide spread distribution of iron in lamprey tissues and additional evidence for the potential value of lampreys for the study of iron metabolism in vertebrates.

We determined whole-fish total mercury (Hg) concentrations of 40 male and 40 female adult sea lampreys (Petromyzon marinus) captured in the Cheboygan River, a tributary to Lake Huron, during May 2011. In addition, bioenergetics modeling was used to explore the effects of sex-related differences in activity and resting (standard) metabolic rate (SMR) on mercury accumulation. The grand mean for Hg concentrations was 519 ng/g (standard error of the mean=46 ng/g). On average, males were 16% higher in Hg concentration than females. Bioenergetics modeling results indicated that 14% higher activity and SMR in males would account for this observed sex difference in Hg concentrations. We concluded that the higher Hg concentration in males was most likely due to higher rate of energy expenditure in males, stemming from greater activity and SMR. Our findings have implications for estimating the effects of sea lamprey populations on mercury cycling within ecosystems, as well as for the proposed opening of sea lamprey fisheries. Eventually, our results may prove useful in improving control of sea lamprey, a pest responsible for substantial damage to fisheries in lakes where it is not native.

We determined whole-fish total mercury (Hg) concentrations of 40 male and 40 female adult sea lampreys (Petromyzon marinus) captured in the Cheboygan River, a tributary to Lake Huron, during May 2011. In addition, bioenergetics modeling was used to explore the effects of sex-related differences in activity and resting (standard) metabolic rate (SMR) on mercury accumulation. The grand mean for Hg concentrations was 519 ng/g (standard error of the mean = 46 ng/g). On average, males were 16% higher in Hg concentration than females. Bioenergetics modeling results indicated that 14% higher activity and SMR in males would account for this observed sex difference in Hg concentrations. We concluded that the higher Hg concentration in males was most likely due to higher rate of energy expenditure in males, stemming from greater activity and SMR. Our findings have implications for estimating the effects of sea lamprey populations on mercury cycling within ecosystems, as well as for the proposed opening of sea lamprey fisheries. Eventually, our results may prove useful in improving control of sea lamprey, a pest responsible for substantial damage to fisheries in lakes where it is not native.

A novel bacterial strain, designated Y11T, was isolated from marine sediment at Weihai in China. Comparative analysis of 16S rRNA gene sequences demonstrated that the novel isolate showed highest similarity to Saccharicrinis fermentans DSM 9555T (94.0 %) and Saccharicrinis carchari SS12T (92.7 %). Strain Y11T was a Gram-stain-negative, rod-shaped, non-endospore-forming, yellow-pigmented bacterium and was able to hydrolyse agar weakly. It was catalase-negative, oxidase-positive, facultatively anaerobic and motile by gliding. Optimal growth occurred at 28-30 °C, at pH 7.0-7.5 and in the presence of 2-3 % (w/v) NaCl. The DNA G+C content was 34.4 mol%. The strain contained MK-7 as the prevalent menaquinone. The major cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0 and C15 : 1ω6c. The predominant polar lipids were phosphatidylethanolamine and two unknown lipids. Data from the present polyphasic taxonomic study clearly place the strain as representing a novel species within the genus Saccharicrinis, for which the name Saccharicrinis marinus sp. nov. is proposed. The type strain is Y11T ( = CICC10837T = KCTC42400T).

Two strains of a gram-positively staining, obligately anaerobic, non-spore-forming, rod-shaped bacterium, designated strains 111-13A and 111-35T, were isolated from human faeces. Analysis of the 16S rRNA gene sequences indicated that these strains were members of the Clostridium coccoides rRNA group of organisms. The nearest relatives of the unknown bacterium were Eubacterium formicigenerans (having a sequence similarity of 94%) and an uncultured bacterium (similarity > 99%). Characterization studies indicated that the unidentified faecal bacterium was biochemically distinct from Eubacterium formicigenerans, members of the Clostridium coccoides group and all other described Eubacterium species. On the basis of the data from these studies, it is proposed that the hitherto unknown rod-shaped bacterium be designated a species of a novel genus, namely Dorea longicatena gen. nov., sp. nov., and that Eubacterium formicigenerans be transferred to this genus as Dorea formicigenerans gen. nov., comb. nov.

Perkinsus marinus, a pathogen of the eastern oyster Crassostrea virginica, is transmitted directly among oysters. Previous studies found viable P. marinus parasites in the feces and pseudofeces of oysters within hours of injection with parasites, suggesting that the parasite ...

The mangrove oyster Saccostrea palmula coexists with the pleasure oyster Crassostrea corteziensis in coastal lagoons of northwest Mexico. Recent discovery of Perkinsus marinus infecting the pleasure oyster in the region prompted evaluation of S. palmula as an alternative P. marinus host. An analysis to determine the possible presence of P. marinus in natural and cultured populations of S. palmula at four coastal lagoons in Sinaloa, Mexico was carried out during October-November 2010. Tissues from apparently healthy S. palmula were evaluated using Ray's fluid thioglycollate method (RFTM), which revealed a Perkinsus sp. to be present in all four locations at 6.7-20.0% prevalence. Histopathological analysis of these specimens showed tissue alterations and parasite forms consistent with moderate P. marinus infection, which was confirmed by ribosomal non-transcribed spacer (NTS)-based PCR assays on DNA samples from oysters positive by RFTM and histology. DNA sequencing of amplified NTS fragments (307 bp) produced a sequence 98-100% similar to GenBank-deposited sequences of the NTS from P. marinus. Fluorescent in situ hybridization for Perkinsus spp. and P. marinus corroborated the PCR results, showing clear hybridization of P. marinus in host tissues. This is the first record of P. marinus infecting a species from genus Saccostrea and the first record of the parasite from coastal lagoons in Sinaloa, Mexico.

Background Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. Results The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. Conclusion The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced - Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

The cellulose-binding proteins, CBPA and CBPB, of rumen cellulolytic bacterium Eubacterium cellulosolvens 5 were biochemically characterized, and their properties were compared. Recombinant CBPA and CBPB were a typical 1,4-beta-endoglucanase. Both proteins bound to insoluble polysaccharides such as Avicel cellulose, acid swollen cellulose, lichenan, chitin, and oat spelt xylan. On the other hand, only recombinant CBPB bound to agarose and starch.

1. Intracellular responses to flashes and steps of light have been recorded from the outer segment and the cell body of rods in the retina of the Bufo marinus. The identification of the origin of recorded responses has been confirmed by intracellular marking. 2. Responses to flashes delivered in darkness or superimposed on a background were analysed. Responses recorded from outer segments conform to the principle of `spectral univariance'. The shape of the response is not affected by enlarging the spot diameter from 150 to 1000 μm. 3. The membrane potential measured in darkness at the outer segments varied from -15 to -25 mV. Injection of steady hyperpolarizing currents increases the size of the response to light; depolarizing currents reduce the response. The mean value of the input resistance is 97 ± 30 MΩ in darkness and increases by 20-30% during illumination. 4. The responses obtained from the cell body of rods have the same shape, time course and spectral sensitivity of those recorded at the outer segment. Injection of steady current at the cell body produces different effects than at the outer segment: hyperpolarizing currents reduce the amplitude of the response to light; depolarizing currents increase the response. 5. The experimental data are fitted according to a model similar to that used to describe the responses of turtle cones (Baylor & Hodgkin, 1974; Baylor, Hodgkin & Lamb, 1974a, b). 6. The model reproduces the electrical responses of the rod outer segment to a variety of stimuli: (a) brief flashes and steps of light in dark adapted conditions; (b) bright flashes superimposed on background illuminations; (c) pairs of flashes delivered at different time intervals. Responses to hyperpolarizing steps of current are also reproduced by the model. ImagesABCD PMID:406383

A new genus, Mogibacterium, is proposed for anaerobic, non-spore-forming, Gram-positive, rod-shaped bacteria which have been isolated from the periodontal pockets of adult human patients with periodontal disease and infected root canals. The novel isolates, strains D2-18T, BA11a-f and D5-2T, were inert in most of the conventional biochemical tests and phenotypically resemble asaccharolytic Eubacterium species. The protein profiles of whole cells on SDS-PAGE gels and Western immunoblotting reaction analysis distinguished these organisms from type strains belonging to the previously described Eubacterium species. The G + C content of the DNA is 45-46 mol% for Mogibacterium pumilum and 46 mol% for Mogibacterium vescum. The levels of DNA-DNA relatedness of these new species to other Eubacterium species, including Eubacterium limosum, Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium saphenum, and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The DNA-DNA hybridization value between M. pumilum and M. vescum was 30%. Eubacterium timidum exhibited DNA homologies with Mogibacterium species which were low (17 and 18%) but clearly higher than with all the other Eubacterium species. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the closest phylogenetic neighbour of Mogibacterium species was E. timidum, and that these three species represent a novel lineage distinct from the previously described genera of Gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, it is also proposed that E. timidum is transferred to the genus Mogibacterium gen. nov. as Mogibacterium timidum gen. nov., comb. nov. (type strain ATCC 33093T).

We report a noteworthy case of Eubacterium plautii infection after kidney transplantation. Our 33-yr-old transplant recipient received standard care; his post-transplant course was uneventful. However, on day 44 he underwent an emergency laparotomy for perforation of the ileum. He was initially treated with ceftazidime, fluconazole and metronidazole, but his fever persisted, so he was switched to meropenem and vancocin. We could not find any cause for his infection. On day 70, his temperature normalized. On day 75, he developed severe leukopenia (280 cell/mL). His cytomegalovirus-DNA test result was negative, so all immunosuppressants, except for prednisone, were stopped; instead, antibiotic prophylaxis was started, using caspofungin, trimethoprim-sulfamethoxazole and ciprofloxacin. On day 83, he underwent percutaneous drainage of massive left pleural effusion. We repeatedly cultured the pleural liquid, but it was not till three wk later that we were finally able to identify the causative organism. We hypothesize that the microorganism - which normally resides on the surface of the intestinal lumen - entered the bloodstream via bacterial translocation, eventually colonizing the pleurae. This translocation was favored by our patient poor clinical condition, his immunosuppressive treatment and his heavy antibiotherapy. Our experience highlights the need for wiser use of antibiotics in transplant recipients.

Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute-binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, whereas the membrane-associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute-binding proteins display a range of glycan-binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6-branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch-degrading Clostridium cluster XIVa organisms in the human gut.

The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from Eubacterium rectale (ErTadZ), in complex with ATP and Mg(2+) , was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker-A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down 'active site'. Homology modelling of the N-terminal atypical receiver domain of CpaE indicates that it has a conserved protein-protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.

Our previous work showed that the adduct between beta-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism.

Methoxychlor [1,1,1-trichloro-2,2-bis(p-methoxyphenyl)ethane], a substitute for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), is a compound of environmental concern because of potential long-term health risks related to its endocrine-disrupting and carcinogenic potency. In order to determine the metabolic fate of methoxychlor and DDT in the human intestinal gut, Eubacterium limosum (ATCC 8486), a strict anaerobe isolated from the human intestine that is capable of O-demethylation toward O-methylated isoflavones, was used as a model intestinal microbial organism. Under anaerobic incubation conditions, E. limosum completely transformed methoxychlor and DDT in 16 days. Based on gas chromatography-mass chromatography analyses, the metabolites produced from methoxychlor and DDT by E. limosum were confirmed to be 1,1-dichloro-2,2-bis(p-methoxyphenyl)ethane (methoxydichlor) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), respectively. This study suggests that E. limosum in the human intestinal gut might be a participant in the reductive dechlorination of methoxychlor to the more antiandrogenic active methoxydichlor.

Summary Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute-binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, while the membrane associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute-binding proteins display a range of glycan-binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6-branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch-degrading Clostridium cluster XIVa organisms in the human gut. PMID:25388295

Perkinsus marinus (Phylum Perkinsozoa), a protozoan parasite of oysters, is considered one of the earliest diverging groups of the lineage leading to dinoflagellates. Perkinsus trophozoites are phagocytosed by oyster hemocytes, where they are likely exposed to reactive oxygen species. As part of its reactive oxygen detoxifying pathway, P. marinus possesses two iron-cofactored SOD (PmSOD1 and PmSOD2). Immunoflourescence analysis of P. marinus trophozoites and gene complementation in yeast revealed that PmSOD1 is targeted to the mitochondria. Surprisingly, although PmSOD2 is characterized by a bipartite N-terminus extension typical of plastid targeting, in preliminary immunofluorescence studies it was visualized as punctuate regions in the cytoplasm that could not be assigned to any organelle. Here, we used immunogold electron microscopy to examine the subcellular localization PmSOD2 in P. marinus trophozoites. Gold grains were mostly associated with single-membrane vesicle-like structures, and eventually, localized to electron-dense, apparently amorphous material present in the lumen of a larger, unique compartment. The images suggested that PmSOD2 is targeted to small vesicles that fuse and/or discharge their content into a larger compartment, possibly the large vacuole typical of the mature trophozoites. In light of the in silico targeting prediction, the association of PmSOD2 with single-membrane compartments raises interesting questions regarding its organellar targeting, and the nature of a putative relic plastid in Perkinsus species.

Population genetic analysis of genotypes comprised of seven microsatellite loci revealed clonal genetic patterns in each of four populations of the protistan estuarine parasite Perkinsus marinus. Each locus was amplified directly from DNA extracted from infected oysters collected from four geographi...

Induction of androgenesis and gynogenesis by applying a pressure (PS) or heat shock (HS) to double the haploid chromosomal set results in progenies possessing only chromosomes from a single parent. This has never been accomplished in representatives of Agnatha. The objective of this study was to induce gynogenesis and androgenesis in sea lamprey Petromyzon marinus. For gynogenesis experiments, ultraviolet (UV)-irradiated sperm was used to activate sea lamprey eggs and HS or PS were applied to inhibit the second meiotic division and consequently induce diploidy in the embryos. The UV irradiation of immobilized sperm was performed for 1 min at 1,719 J m-2. HS of 35 ?? 1??C for 2 min and PS of 9,000 psi for 4 min were applied at different times after egg activation (8, 12, 20, and 24 min or 8, 16, and 24 min for HS or PS, respectively). Regardless of the induction time of the HS, survivals at pre-hatching stage were similar. In contrast, PS applied 8 min after activation appears to increase survival rate of pre-hatched embryos in comparison to 16 and 24 min after activation. In control groups, without shock treatment (no diploidization), there were no survivors. All deformed, gynogenetic embryos were confirmed to be haploids and died prior to burying themselves in the sand. We confirmed by flow cytometry that progenies produced using both shock methods surviving to the next stage, burying in the substrate, were diploid gynogenetic. For the androgenesis experiments, UV-irradiated eggs (1,719 J m-2 for 1 min) were fertilized with non-treated sperm and HS was applied to restore diploidy of the eggs. Several attempts have been made to optimize the parameters used. HS of 35 ?? 1??C was applied 110, 140, 170, 200, and 230 min after activation for 2 min. Low yields of androgens were obtained and all animals died within a week after hatching. These techniques will allow to establish meiotic gynogenetic lines of sea lamprey for determining sex differentiation in this species

Species of Perkinsus are responsible for high mortalities of bivalve molluscs world-wide. Techniques to accurately estimate parasites in tissues are required to improve understanding of perkinsosis. This study quantifies the number and tissue distribution of Perkinsus marinus in ...

The progression of diseases caused by the oyster parasites, Perkinsus marinus and Haplosporidium nelsoni, were evaluated by periodic sampling (May 1994 - December 1995) of oysters, Crassostrea virginica, on an artificial reef located in the Piankatank River, Virginia. The infecti...

Reindeer are able to eat and utilize lichens as an important source of energy and nutrients. In the current study, the activities of antibiotic secondary metabolites including usnic, antranoric, fumarprotocetraric, and lobaric acid commonly found in lichens were tested against a collection of 26 anaerobic rumen bacterial isolates from reindeer ( Rangifer tarandus tarandus) using the agar diffusion method. The isolates were identified based on their 16S ribosomal ribonucleic acid (rRNA) gene sequences. Usnic acid had a potent antimicrobial effect against 25 of the isolates, belonging to Clostridiales, Enterococci, and Streptococci. Isolates of Clostridia and Streptococci were also susceptible to atranoric and lobaric acid. However, one isolate (R3_91_1) was found to be resistant to usnic, antranoric, fumarprotocetraric, and lobaric acid. R3_91_1 was also seen invading and adhering to lichen particles when grown in a liquid anaerobic culture as demonstrated by transmission electron microscopy. This was a Gram-negative, nonmotile rod (0.2-0.7 × 2.0-3.5 μm) with a deoxyribonucleic acid G + C content of 47.0 mol% and main cellular fatty acids including 15:0 anteiso-dimethyl acetal (DMA), 16:0 iso-fatty acid methyl ester (FAME), 13:0 iso-3OH FAME, and 17:0 anteiso-FAME, not matching any of the presently known profiles in the MIDI database. Combined, the phenotypic and genotypic traits including the 16S rRNA gene sequence show that R3_91_1 is a novel species inside the order Clostridiales within the family Lachnospiraceae, for which we propose the name Eubacterium rangiferina. This is the first record of a rumen bacterium able to tolerate and grow in the presence of usnic acid, indicating that the rumen microorganisms in these animals have adapted mechanisms to deal with lichen secondary metabolites, well known for their antimicrobial and toxic effects.

The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (+)-taxifolin but not that of (-)-taxifolin. The Km for (+)-taxifolin was 6.4 ± 0.8 μM, and the Vmax was 108 ± 4 μmol min(-1) (mg protein)(-1) The enzyme also isomerized (+)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439-1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus IMPORTANCE: Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria.

Reindeer are able to eat and utilize lichens as an important source of energy and nutrients. In the current study, the activities of antibiotic secondary metabolites including usnic, antranoric, fumarprotocetraric, and lobaric acid commonly found in lichens were tested against a collection of 26 anaerobic rumen bacterial isolates from reindeer (Rangifer tarandus tarandus) using the agar diffusion method. The isolates were identified based on their 16S ribosomal ribonucleic acid (rRNA) gene sequences. Usnic acid had a potent antimicrobial effect against 25 of the isolates, belonging to Clostridiales, Enterococci, and Streptococci. Isolates of Clostridia and Streptococci were also susceptible to atranoric and lobaric acid. However, one isolate (R3_91_1) was found to be resistant to usnic, antranoric, fumarprotocetraric, and lobaric acid. R3_91_1 was also seen invading and adhering to lichen particles when grown in a liquid anaerobic culture as demonstrated by transmission electron microscopy. This was a Gram-negative, nonmotile rod (0.2-0.7 x 2.0-3.5 microm) with a deoxyribonucleic acid G + C content of 47.0 mol% and main cellular fatty acids including 15:0 anteiso-dimethyl acetal (DMA), 16:0 iso-fatty acid methyl ester (FAME), 13:0 iso-3OH FAME, and 17:0 anteiso-FAME, not matching any of the presently known profiles in the MIDI database. Combined, the phenotypic and genotypic traits including the 16S rRNA gene sequence show that R3_91_1 is a novel species inside the order Clostridiales within the family Lachnospiraceae, for which we propose the name Eubacterium rangiferina. This is the first record of a rumen bacterium able to tolerate and grow in the presence of usnic acid, indicating that the rumen microorganisms in these animals have adapted mechanisms to deal with lichen secondary metabolites, well known for their antimicrobial and toxic effects.

Acetogens are a specialized group of anaerobic bacteria able to produce acetate from CO2 and H2 via the Wood-Ljungdahl pathway. In some gut environments acetogens can compete with methanogens for H2, and as a result rumen acetogens are of interest in the development of microbial approaches for methane mitigation. The acetogen Eubacterium limosum SA11 was isolated from the rumen of a New Zealand sheep and its genome has been sequenced to examine its potential application in methane mitigation strategies, particularly in situations where hydrogenotrophic methanogens are inhibited resulting in increased H2 levels in the rumen. The 4.15 Mb chromosome of SA11 has an average G + C content of 47 %, and encodes 3805 protein-coding genes. There is a single prophage inserted in the chromosome, and several other gene clusters appear to have been acquired by horizontal transfer. These include genes for cell wall glycopolymers, a type VII secretion system, cell surface proteins and chemotaxis. SA11 is able to use a variety of organic substrates in addition to H2/CO2, with acetate and butyrate as the principal fermentation end-products, and genes involved in these metabolic pathways have been identified. An unusual feature is the presence of 39 genes encoding trimethylamine methyltransferase family proteins, more than any other bacterial genome. Overall, SA11 is a metabolically versatile organism, but its ability to grow on such a wide range of substrates suggests it may not be a suitable candidate to take the place of hydrogen-utilizing methanogens in the rumen.

An experimental mesocosm study suggested larval sea lamprey Petromyzon marinus detect and respond to an alarm cue released by dead adult conspecifics. Larvae exhibited a reduced tendency to move downstream when exposed to the cue and were less likely to move under continuous v. pulsed exposure. These findings support the hypothesis that short-term exposure to the alarm cue would probably result in retraction into the burrow, consistent with the blind, cryptic lifestyle of the larval P. marinus.

The cane or marine toad (Chaunus marinus, formerly Bufo marinus) was introduced to the Northern Mariana Islands starting in the 1930s. The effects of this exotic predator on native vertebrates (especially lizards) are largely unknown. We analysed the stomach contents of 336 cane toads collected from the island of Rota, with the goal of estimating the level of toad predation on native vertebrates. Beetles, ants, millipedes, and grasshoppers/crickets comprised the majority of prey classes consumed by toads. The introduced Brahminy blindsnake (Ramphotyphlops braminus; N = 6) and conspecific cane toads (N = 4) were the vertebrates most commonly found in toad stomachs. Skinks (Emoia; N = 2) were the only native vertebrates represented in our sample. The small numbers of nocturnal terrestrial vertebrates native to Rota likely translates to relatively low rates of predation by cane toads on native vertebrates.

Dermo disease in the eastern oyster (Crassostrea virginica) is caused by an intracellular protistan parasite Perkinsus marinus. The progression and outcome of this disease is determined by a complex interplay between the host's immunity and parasite's escape mechanisms, both of which can be influenced by environmental pollutants including heavy metals such as copper (Cu). The goal of the present study was to determine the effects of Cu on the levels of apoptosis (which can serve as an important host defense mechanism) in oyster immune cells (hemocytes) in vitro and in vivo as well as on the establishment of P. marinus infections in vivo. Surprisingly, Cu exerted opposing effects on apoptosis levels of hemocytes in vitro and in vivo, stimulating apoptosis in isolated hemocytes but suppressing it during Cu exposure of whole oysters. The mechanisms of this effect are presently unknown and may be related to the different bioavailability of the metal in vitro and in vivo. As expected, Cu accumulated in oyster soft tissues during in vitro exposure. Unexpectedly, this metal also strongly accumulated in hemolymph plasma which is classically considered isoionic with the surrounding seawater, likely reflecting the presence of soluble Cu-binding proteins in oyster plasma. Cu reduced growth of P. marinus in vitro and greatly reduced infection levels of hemocytes in vivo, presumably by direct toxic effects on the parasite. As a possible parasitic counterbalance, Cu accumulation in the hemocytes was reduced by P. marinus infection, although this reduction was not sufficient to prevent the parasiticidal effects of the heavy metal in vivo. This effect of Cu may be useful as a potential therapeutic against Dermo disease in aquaculture conditions. Overall, this study provides important new insights into the potential role of environmental metals in host-parasite relationships and disease dynamics in C. virginica.

Aplochiton is a small genus of galaxiid fishes endemic to Patagonia and the Falkland Islands whose taxonomy is insufficiently resolved. Recent genetic analyses confirmed the existence of only two closely related species, Aplochiton taeniatus and Aplochiton zebra, while a third controversial species, Aplochiton marinus, remained lost to synonymy with A. taeniatus. Using an integrative taxonomy framework, we studied original samples and published sequences from a broad range in western Patagonia and the Falkland Islands, and generated robust species hypotheses based on single-locus (Cytochrome Oxidase subunit I; COI) species-delineation methods and known diagnostic morphological characters analyzed in a multivariate context. Results revealed three distinct evolutionary lineages that morphologically resemble, in important respects, existing nominal species descriptions. Interestingly, the lineage associated with A. marinus was unambiguously identifiable (100% accuracy) both from the genetic and morphological viewpoints. In contrast, the morphology of A. taeniatus and A. zebra overlapped substantially, mainly due to the high variability of A. taeniatus. Discriminant function analysis aided the identification of these species with 83.9% accuracy. Hence, for their unambiguous identification, genetic screening is needed. A. marinus has seldom been documented, and when recorded, it has always been found in sites with clear marine influence. It is possible that only A. marinus preserves a life cycle related to the sea akin to the hypothesized ancestral galaxiid. We did not find evidence of claimed diadromy in A. taeniatus or A. zebra, and, therefore, these should be regarded as freshwater species. Finally, a lack of phylogeographic patterns and overrepresentation of uncommon haplotypes suggested demographic expansions in recent evolutionary time, especially of A. zebra, in line with the hypothesis of large-scale range expansion and lineage spread in western Patagonia.

Aplochiton is a small genus of galaxiid fishes endemic to Patagonia and the Falkland Islands whose taxonomy is insufficiently resolved. Recent genetic analyses confirmed the existence of only two closely related species, Aplochiton taeniatus and Aplochiton zebra, while a third controversial species, Aplochiton marinus, remained lost to synonymy with A. taeniatus. Using an integrative taxonomy framework, we studied original samples and published sequences from a broad range in western Patagonia and the Falkland Islands, and generated robust species hypotheses based on single-locus (Cytochrome Oxidase subunit I; COI) species-delineation methods and known diagnostic morphological characters analyzed in a multivariate context. Results revealed three distinct evolutionary lineages that morphologically resemble, in important respects, existing nominal species descriptions. Interestingly, the lineage associated with A. marinus was unambiguously identifiable (100% accuracy) both from the genetic and morphological viewpoints. In contrast, the morphology of A. taeniatus and A. zebra overlapped substantially, mainly due to the high variability of A. taeniatus. Discriminant function analysis aided the identification of these species with 83.9% accuracy. Hence, for their unambiguous identification, genetic screening is needed. A. marinus has seldom been documented, and when recorded, it has always been found in sites with clear marine influence. It is possible that only A. marinus preserves a life cycle related to the sea akin to the hypothesized ancestral galaxiid. We did not find evidence of claimed diadromy in A. taeniatus or A. zebra, and, therefore, these should be regarded as freshwater species. Finally, a lack of phylogeographic patterns and overrepresentation of uncommon haplotypes suggested demographic expansions in recent evolutionary time, especially of A. zebra, in line with the hypothesis of large-scale range expansion and lineage spread in western Patagonia. PMID

Mercury compounds pollute many aquatic habitats and are extremely toxic to aquatic organisms. Acute toxicity of waterborne methyl mercury has been studied in several teleost species. Lampreys are taxonomically distant from teleosts and are used for comparative toxicological purposes. Landlocked sea lampreys, Petromyzon marinus, inhabit the Great Lakes region, and their larvae (ammocoetes) burrow in stream sediments. In this study, the authors present toxicity curves for ammocoetes exposed acutely to methyl mercuric chloride solutions. Susceptibility was related to temperature and animal size.

Culture of the pleasure oyster Crassostrea corteziensis is emerging as an alternative to the Pacific oyster (Crassostrea gigas) for oyster producers, who face severe mortalities since 1997 in Northwest México. For determining the health status of this species, we conducted a histopathological analysis of cultured populations from two estuaries in the Pacific coast of México. Macroscopical analysis revealed animals with transparent and retracted mantle. Histopathological analysis of these specimens showed tissue alterations and parasitic forms consistent with Perkinsus sp. infection. Stages of the parasite identified included tomont and trophozoites with an eccentric vacuole characteristic of Perkinsus spp. Pieces of tissues of infected oysters were incubated in Fluid Thioglycollate Medium (FTM) resulting in blue-black hypnospores after incubation. The identity of the parasite was confirmed by species specific PCR-based assay in DNA samples from oysters, tissue fractions from FTM cultures, and deparaffined samples with Perkinsus-like parasite detected by histology. Sequencing of positive amplified fragments (307bp) showed a sequence similar to Perkinsus marinus strain TXsc NTS ribosomal RNA gene (100% coverage and 98% identity, GenBank Accession No. AF497479.1) and to P. marinus, Genomic DNA, (100% coverage and 97% identity, GenBank Accession No. S78416.1). The prevalence of P. marinus varied from 1 to 5% in Boca del Camichín and from 1 to 6% in Pozo Chino. In general, the intensity of infection was moderate. The infection was observed in oysters from 31 to 110mm of shell length. This is the first record of P. marinus in oysters from the North America Pacific coast and the first record in C. corteziensis. The origin of this parasite in the area is unknown, but it may be associated to introductions of Crassostrea virginica from the East coast of United States of America or Gulf of México.

Cellobiose 2-epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44-54% identity to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 degrees C and pH 7.0-8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4-O-beta-D-galactopyranosyl-D-mannose).

Fucosyllactoses (2′- or 3′-FL) account for up to 20% of human milk oligosaccharides (HMOs). Infant bifidobacteria, such as Bifidobacterium longum subsp. infantis, utilize the lactose moiety to form lactate and acetate, and metabolize L-fucose to 1,2-propanediol (1,2-PD). Eubacterium hallii is a common member of the adult gut microbiota that can produce butyrate from lactate and acetate, and convert 1,2-PD to propionate. Recently, a Swiss cohort study identified E. hallii as one of the first butyrate producers in the infant gut. However, the global prevalence of E. hallii and its role in utilization of HMO degradation intermediates remains unexplored. Fecal 16S rRNA gene libraries (n = 857) of humans of all age groups from Venezuela, Malawi, Switzerland, and the USA were screened for the occurrence of E. hallii. Single and co-culture experiments of B. longum subsp. infantis and E. hallii were conducted in modified YCFA containing acetate and glucose, L-fucose, or FL. Bifidobacterium spp. (n = 56) of different origin were screened for the ability to metabolize L-fucose. Relative abundance of E. hallii was low (10−5–10−3%) during the first months but increased and reached adult levels (0.01–10%) at 5–10 years of age in all four populations. In single culture, B. longum subsp. infantis grew in the presence of all three carbohydrates while E. hallii was metabolically active only with glucose. In co-culture E. hallii also grew with L-fucose or FL. In co-cultures grown with glucose, acetate, and glucose were consumed and nearly equimolar proportions of formate and butyrate were formed. B. longum subsp. infantis used L-fucose and produced 1,2-PD, acetate and formate in a ratio of 1:1:1, while 1,2-PD was used by E. hallii to form propionate. E. hallii consumed acetate, lactate and 1,2-PD released by B. longum subsp. infantis from FL, and produced butyrate, propionate, and formate. Beside B. longum subsp. infantis, Bifidobacterium breve, and a strain of B

Fucosyllactoses (2'- or 3'-FL) account for up to 20% of human milk oligosaccharides (HMOs). Infant bifidobacteria, such as Bifidobacterium longum subsp. infantis, utilize the lactose moiety to form lactate and acetate, and metabolize L-fucose to 1,2-propanediol (1,2-PD). Eubacterium hallii is a common member of the adult gut microbiota that can produce butyrate from lactate and acetate, and convert 1,2-PD to propionate. Recently, a Swiss cohort study identified E. hallii as one of the first butyrate producers in the infant gut. However, the global prevalence of E. hallii and its role in utilization of HMO degradation intermediates remains unexplored. Fecal 16S rRNA gene libraries (n = 857) of humans of all age groups from Venezuela, Malawi, Switzerland, and the USA were screened for the occurrence of E. hallii. Single and co-culture experiments of B. longum subsp. infantis and E. hallii were conducted in modified YCFA containing acetate and glucose, L-fucose, or FL. Bifidobacterium spp. (n = 56) of different origin were screened for the ability to metabolize L-fucose. Relative abundance of E. hallii was low (10(-5)-10(-3)%) during the first months but increased and reached adult levels (0.01-10%) at 5-10 years of age in all four populations. In single culture, B. longum subsp. infantis grew in the presence of all three carbohydrates while E. hallii was metabolically active only with glucose. In co-culture E. hallii also grew with L-fucose or FL. In co-cultures grown with glucose, acetate, and glucose were consumed and nearly equimolar proportions of formate and butyrate were formed. B. longum subsp. infantis used L-fucose and produced 1,2-PD, acetate and formate in a ratio of 1:1:1, while 1,2-PD was used by E. hallii to form propionate. E. hallii consumed acetate, lactate and 1,2-PD released by B. longum subsp. infantis from FL, and produced butyrate, propionate, and formate. Beside B. longum subsp. infantis, Bifidobacterium breve, and a strain of B. longum subsp

3-Methylitaconate-Delta-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K(m) but > 1000-fold lower k(cat). The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 A. The asymmetric unit of the P2(1)2(1)2(1) unit cell with parameters a = 53.1 A, b = 142.3 A, and c = 228.4 A contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an alpha/beta-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Delta-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as

Eubacterium hallii is considered an important microbe in regard to intestinal metabolic balance due to its ability to utilize glucose and the fermentation intermediates acetate and lactate, to form butyrate and hydrogen. Recently, we observed that E. hallii is capable of metabolizing glycerol to 3-hydroxypropionaldehyde (3-HPA, reuterin) with reported antimicrobial properties. The key enzyme for glycerol to 3-HPA conversion is the cobalamin-dependent glycerol/diol dehydratase PduCDE which also utilizes 1,2-propanediol (1,2-PD) to form propionate. Therefore our primary goal was to investigate glycerol to 3-HPA metabolism and 1,2-PD utilization by E. hallii along with its ability to produce cobalamin. We also investigated the relative abundance of E. hallii in stool of adults using 16S rRNA and pduCDE based gene screening to determine the contribution of E. hallii to intestinal propionate formation. We found that E. hallii utilizes glycerol to produce up to 9 mM 3-HPA but did not further metabolize 3-HPA to 1,3-propanediol. Utilization of 1,2-PD in the presence and absence of glucose led to the formation of propanal, propanol and propionate. E. hallii formed cobalamin and was detected in stool of 74% of adults using 16S rRNA gene as marker gene (n = 325). Relative abundance of the E. hallii 16S rRNA gene ranged from 0 to 0.59% with a mean relative abundance of 0.044%. E. hallii PduCDE was detected in 63 to 81% of the metagenomes depending on which subunit was investigated beside other taxons such as Ruminococcus obeum, R. gnavus, Flavonifractor plautii, Intestinimonas butyriciproducens, and Veillonella spp. In conclusion, we identified E. hallii as a common gut microbe with the ability to convert glycerol to 3-HPA, a step that requires the production of cobalamin, and to utilize 1,2-PD to form propionate. Our results along with its ability to use a broad range of substrates point at E. hallii as a key species within the intestinal trophic chain with the potential to

Eubacterium hallii is considered an important microbe in regard to intestinal metabolic balance due to its ability to utilize glucose and the fermentation intermediates acetate and lactate, to form butyrate and hydrogen. Recently, we observed that E. hallii is capable of metabolizing glycerol to 3-hydroxypropionaldehyde (3-HPA, reuterin) with reported antimicrobial properties. The key enzyme for glycerol to 3-HPA conversion is the cobalamin-dependent glycerol/diol dehydratase PduCDE which also utilizes 1,2-propanediol (1,2-PD) to form propionate. Therefore our primary goal was to investigate glycerol to 3-HPA metabolism and 1,2-PD utilization by E. hallii along with its ability to produce cobalamin. We also investigated the relative abundance of E. hallii in stool of adults using 16S rRNA and pduCDE based gene screening to determine the contribution of E. hallii to intestinal propionate formation. We found that E. hallii utilizes glycerol to produce up to 9 mM 3-HPA but did not further metabolize 3-HPA to 1,3-propanediol. Utilization of 1,2-PD in the presence and absence of glucose led to the formation of propanal, propanol and propionate. E. hallii formed cobalamin and was detected in stool of 74% of adults using 16S rRNA gene as marker gene (n = 325). Relative abundance of the E. hallii 16S rRNA gene ranged from 0 to 0.59% with a mean relative abundance of 0.044%. E. hallii PduCDE was detected in 63 to 81% of the metagenomes depending on which subunit was investigated beside other taxons such as Ruminococcus obeum, R. gnavus, Flavonifractor plautii, Intestinimonas butyriciproducens, and Veillonella spp. In conclusion, we identified E. hallii as a common gut microbe with the ability to convert glycerol to 3-HPA, a step that requires the production of cobalamin, and to utilize 1,2-PD to form propionate. Our results along with its ability to use a broad range of substrates point at E. hallii as a key species within the intestinal trophic chain with the potential to

Prochlorococcus is the smallest known oxygenic phototrophic marine cyanobacterium dominating the mid-latitude oceans. Physiologically and genetically distinct P. marinus isolates from many oceans in the world were assigned two different groups, a tightly clustered high-light (HL)-adapted and a divergent low-light (LL-) adapted clade. Phylogenetic analysis of this cyanobacterium on the basis of 16S rRNA and other conserved genes did not show consistency with its phenotypic behavior. We analyzed phylogeny of this genus on the basis of complete genome sequences through genome alignment, overlapping-gene content and gene-order approach. Phylogenetic tree of P. marinus obtained by comparing whole genome sequences in contrast to that based on 16S rRNA gene, corresponded well with the HL/LL ecotypic distinction of twelve strains and showed consistency with phenotypic classification of P. marinus. Evidence for the horizontal descent and acquisition of genes within and across the genus was observed. Many genes involved in metabolic functions were found to be conserved across these genomes and many were continuously gained by different strains as per their needs during the course of their evolution. Consistency in the physiological and genetic phylogeny based on whole genome sequence is established. These observations improve our understanding about the adaptation and diversification of these organisms under evolutionary pressure.

Perkinsus marinus is a pathogen responsible for severe mortalities of the eastern oyster Crassostrea virginica along the East and Gulf coasts of the United States. When cultivated, the pathogenicity of this microorganism decreases significantly, hampering the study of its virulence factors. Recent investigations have shown a significant increase of the in vivo virulence of P. marinus exposed to oyster pallial mucus. In the current study, we investigated the effect of pallial mucus on P. marinus gene expression compared with cultures supplemented with oyster digestive extracts or with un-supplemented cultures. In parallel, parasite cells cultured under these three conditions were used to challenge oysters and to assess virulence in vivo. Perkinsus marinus mRNA sequencing was performed on an Illumina GAIIX sequencer and data were analysed using the Tuxedo RNAseq suite for mapping against the draft P. marinus genome and for differential expression analysis. Results showed that exposure of P. marinus to mucus induces significant regulation of nearly 3,600 transcripts, many of which are considered as putative virulence factors. Pallial mucus is suspected to mimic internal host conditions, thereby preparing the pathogen to overcome defense factors before invasion. This hypothesis is supported by significant regulation in several antioxidant proteins, heat shock proteins, protease inhibitors and proteasome subunits. In addition, mucus exposure induced the modulation of several genes known to affect immunity and apoptosis in vertebrates and invertebrates. Several proteases (proteolysis) and merozoite surface proteins (cell recognition) were also modulated. Overall, these results provide a baseline for targeted, in depth analysis of candidate virulence factors in P. marinus.

Sea lampreys (Petromyzon marinus) are members of the ancient class Agnatha and undergo a metamorphosis that transforms blind, sedentary, filter-feeding larvae into free-swimming, parasitic juveniles. Thyroid hormones (THs) appear to be important for lamprey metamorphosis, however, serum TH concentrations are elevated in the larval phase, decline rapidly during early metamorphosis and remain low until metamorphosis is complete; these TH fluctuations are contrary to those of other metamorphosing vertebrates. Moreover, thyroid hormone synthesis inhibitors (goitrogens) induce precocious metamorphosis and exogenous TH treatments disrupt natural metamorphosis in P. marinus. Given that THs exert their effects by binding to TH nuclear receptors (TRs) that often act as heterodimers with retinoid X receptors (RXRs), we cloned and characterized these receptors from P. marinus and examined their expression during metamorphosis. Two TRs (PmTR1 and PmTR2) and three RXRs (PmRXRs) were isolated from P. marinus cDNA. Phylogenetic analyses group the PmTRs together on a branch prior to the gnathostome TRα/β split. The three RXRs also group together, but our data indicated that these transcripts are most likely either allelic variants of the same gene locus, or the products of a lamprey-specific duplication event. Importantly, these P. marinus receptors more closely resemble vertebrate as opposed to invertebrate chordate receptors. Functional analysis revealed that PmTR1 and PmTR2 can activate transcription of TH-responsive genes when treated with nanomolar concentrations of TH and they have distinct pharmacological profiles reminiscent of vertebrate TRβ and TRα, respectively. Also similar to other metamorphosing vertebrates, expression patterns of the PmTRs during lamprey metamorphosis suggest that PmTR1 has a dynamic, tissue-specific expression pattern that correlates with tissue morphogenesis and biochemical changes and PmTR2 has a more uniform expression pattern. This TR

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10(7) cells g (dry weight) of feces(-1). The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.

Single-celled bacterivorous eukaryotes offer excellent test cases for evaluation of the frequency of prey-to-predator lateral gene transfer (LGT). Here we use analysis of expressed sequence tag (EST) data sets to quantify the extent of LGT from eubacteria to two amoebae, Acanthamoeba castellanii and Hartmannella vermiformis. Stringent screening for LGT proceeded in several steps intended to enrich for authentic events while at the same time minimizing the incidence of false positives due to factors such as limitations in database coverage and ancient paralogy. The results were compared with data obtained when the same methodology was applied to EST libraries from a number of other eukaryotic taxa. Significant differences in the extent of apparent eubacterium-to-eukaryote LGT were found between taxa. Our results indicate that there may be substantial inter-taxon variation in the number of LGT events that become fixed even between amoebozoan species that have similar feeding modalities.

Delaware's Inland Bays (DIB), USA, are subject to blooms of potentially harmful raphidophytes, including Heterosigma akashiwo. In 2004, a dense bloom was observed in a low salinity tributary of the DIB. Light microscopy initially suggested that the species was H. akashiwo; however, the cells were smaller than anticipated. 18S rDNA sequences of isolated cultures differed substantially from all raphidophyte sequences in GenBank. Phylogenetic analysis placed it approximately equidistant from Chattonella and Heterosigma with only ~96% sequence homology with either group. Here, we describe this marine raphidophyte as a novel genus and species, Viridilobus marinus (gen. et sp. nov.). We also compared this species with H. akashiwo, because both species are superficially similar with respect to morphology and their ecological niches overlap. V. marinus cells are ovoid to spherical (11.4 × 9.4 μm), and the average number of chloroplasts (4 per cell) is lower than in H. akashiwo (15 per cell). Pigment analysis of V. marinus revealed the presence of fucoxanthin, violaxanthin, and zeaxanthin, which are characteristic of marine raphidophytes within the family Chattonellaceae of the Raphidophyceae. TEM and confocal microscopy, however, revealed diagnostic microscopic and ultrastructural characteristics that distinguish it from other raphidophytes. Chloroplasts were in close association with the nucleus and thylakoids were arranged either parallel or perpendicular to the cell surface. Putative mucocysts were identified, but trichocysts were not observed. These features, along with DNA sequence data, distinguish this species from all other raphidophyte genera within the family Chattonellaceae of the Raphidophyceae.

Antibody-functionalized, Au-gated AlGaN/GaN high electron mobility transistors (HEMTs) were used to detect Perkinsus marinus. The antibody was anchored to the gate area through immobilized thioglycolic acid. The AlGaN/GaN HEMT drain-source current showed a rapid response of less than 5 s when the infected solution was added to the antibody-immobilized surface. The sensor can be recycled with a phosphate buffered saline wash. These results clearly demonstrate the promise of field-deployable electronic biological sensors based on AlGaN/GaN HEMTs for Perkinsus marinus detection.

Mechanical and electromechanical barriers played a significant role in the initial attempts to control sea lamprey (Petromyzon marinus) populations in the upper Great Lakes. More recently electromechanical weirs have been used to assess the relative abundance of spawning-run sea lampreys in Lake Superior. Development of an integrated control approach to sea lamprey control has stimulated an ongoing research program to define structural and/or velocity criteria that can be used to design barrier dams that block spawning runs of sea lamprey

Limnothrix (strain AC0243) is a cyanobacterium, which has only recently been identified as toxin producing. Under laboratory conditions, Bufo marinus larvae were exposed to 100,000 cells mL−1 of Limnothrix (strain AC0243) live cultures for seven days. Histological examinations were conducted post mortem and revealed damage to the notochord, eyes, brain, liver, kidney, pancreas, gastrointestinal tract, and heart. The histopathological results highlight the toxicological impact of this strain, particularly during developmental stages. Toxicological similarities to β-N-Methylamino-l-alanine are discussed. PMID:24662524

Levels of estradiol, progesterone, and testosterone were determined in plasma of sea lamprey (Petromyzon marinus) undergoing certain behaviors associated with spawning in natural and artifical stream environments. Significantly higher levels of estradiol, progesterone, and testosterone were found in males than in females. In the artifical spawning channel, levels of estradiol were significantly higher in females exhibiting resting and swimming behaviors than in fanning, nest building, and spawning behaviors. No significant correlation was found with either progesterone or testosterone levels and the various reproductive behaviors. The data presented are the first experimental evidence that suggest gonadal steroids may be correlated with certain reproductive behaviors in the sea lamprey.

Lampreys represent one of the most ancient vertebrate lineages enclosing a special interest for genetic and epigenetic studies. The sea lamprey (Petromyzon marinus) is an anadromous species that experiences metamorphosis all the way up to the adult stage. Although representing a gradual process, metamorphosis in this species involves dramatic conversions with regard to physiological together with structural body changes preparing individuals for a marine and parasitic life; in consequence, multiple gene expression modifications are expected. The implications of thyroid hormones and HOX gene expression changes have previously been reported in this species and also in other vertebrate species. Nonetheless, information lacks on how these genes are regulated in lampreys. We here report about the existence of methylation pattern differences between the adult and the larvae sea lamprey life cycle stages making use of the Methylation-Sensitive Amplified Polymorphism (MSAP) technique. Differentially methylated fragment sequencing allowed to establish homologous identities with HOX genes involved in morphogenesis, along with genes related to the water balance and to the osmotic homoeostasis, all associated to a marine environment adaptation. These results provide evidences revealing that DNA methylation plays a role in the epigenetic regulation of the P. marinus post-natal development representing a starting point for future studies. To the best of our knowledge, this is the first study which detects DNA methylation changes associated with metamorphosis in lampreys.

Heterotrophic lineages of Heterokonta (or stramenopiles), in contrast to a single monophyletic group of autotrophs, Ochrophyta, form several clades that independently branch off the heterokont stem lineage. The nearest neighbors of Ochrophyta in the phylogenetic tree appear to be almost exclusively bacterivorous, whereas the hypothesis of plastid acquisition by the ancestors of the ochrophyte lineage suggests an ability to engulf eukaryotic alga. In line with this hypothesis, the heterotrophic predator at the base of the ochrophyte lineage may be regarded as a model for the ochrophyte ancestor. Here, we present a new genus and species of marine free-living heterotrophic heterokont Develorapax marinus, which falls into an isolated heterokont cluster, along with the marine flagellate Developayella elegans, and is able to engulf eukaryotic cells. Together with environmental sequences D. marinus and D. elegans form a class-level clade Developea nom. nov. represented by species adapted to different environmental conditions and with a wide geographical distribution. The position of Developea among Heterokonta in large-scale phylogenetic tree is discussed. We propose that members of the Developea clade represent a model for transition from bacterivory to a predatory feeding mode by selection for larger prey. Presumably, such transition in the grazing strategy is possible in the presence of bacterial biofilms or aggregates expected in eutrophic environment, and has likely occurred in the ochrophyte ancestor. PMID:27536283

Plant lignans, such as pinoresinol diglucoside, secoisolariciresinol diglucoside and arctiin, are metabolized to mammalian lignans, enterolactone or enterodiol, by human intestinal bacteria. Their metabolic processes include deglucosylation, ring cleavage, demethylation, dehydroxylation and oxidation. Here we isolated an intestinal bacterium capable of demethylating arctigenin, an aglycone of arctiin, to 2,3-bis(3,4-dihydroxybenzyl)butyrolactone (1) from human feces, and identified as an Eubacterium species (E. sp. ARC-2), which is similar to Eubacterium limosum on the basis of morphological and biochemical properties and 16S rRNA gene sequencing. By incubating with E. sp. ARC-2, arctigenin was converted to 1 through stepwise demethylation. Demethylation of arctigenin by E. sp. ARC-2 was tetrahydrofolate- and ATP-dependent, indicating that the reaction was catalyzed by methyltransferase. Moreover, E. sp. ARC-2 transformed secoisolariciresinol to 2,3-bis(3,4-dihydroxybenzyl)-1,4-butanediol by demethylation.

The progression of diseases caused by the oyster parasites, Perkinsus marinus and Haplosporidium nelsoni, were evaluated by periodic sampling (May 1994-Dec. 1995) of oysters, Crassostrea virginica, that set on an artificial reef located in the Piankatank River, Virginia, in Augus...

Field and in vitro studies have shown that high salinities and temperatures promote the proliferation and dissemination of Perkinsus marinus in several environments. In Brazil, the parasite infects native oysters Crassostrea gasar and Crassostrea rhizophorae in the Northeast (NE), where the temperature is high throughout the year. Despite the high prevalence of Perkinsus spp. infection in oysters from the NE of Brazil, no mortality events were reported by oyster farmers to date. The present study evaluated the effects of salinity (5, 20 and 35 psu) and temperature (15, 25 and 35 °C) on in vitro proliferation of P. marinus isolated from a host (C. rhizophorae) in Brazil, for a period of up to 15 days and after the return to the control conditions (22 days; recovery). Different cellular parameters (changes of cell phase's composition, cell density, viability and production of reactive oxygen species) were analysed using flow cytometry. The results indicate that the P. marinus isolate was sensitive to the extreme salinities and temperatures analysed. Only the highest temperature caused lasting cell damage under prolonged exposure, impairing P. marinus recovery, which is likely to be associated with oxidative stress. These findings will contribute to the understanding of the dynamics of perkinsiosis in tropical regions.

A Gram-positive-staining, coccoid-shaped, non-motile, asporogenous, obligately anaerobic and butyrate-producing bacterium was recovered from a healthy human's faeces. The organism was isolated by the enrichment culture technique using yeast extract-casein hydrolysate-fatty acids broth supplemented with 0.5 % mucin. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the novel strain should be classified as a member of the Eubacterium desmolans-related cluster in the family Ruminococcaceae. Furthermore, this analysis demonstrated that the type strains of Butyricicoccus pullicaecorum (95.6 %) and Eubacterium desmolans (94.7 %) were the closest phylogenetic neighbours to strain YIT 12789T. However, DNA‒DNA reassociation values with these closest strains were less than 20 %. On the basis of the phenotypic, genotypic and chemotaxonomic features, the novel coccoid-shaped bacterium should be designated as a representative of a novel species of the genus Butyricicoccus, for which the name Butyricicoccus faecihominis sp. nov. is proposed. The type strain is YIT 12789T (=JCM 31056T=DSM 100989T). It is also proposed that Eubacterium desmolans be reclassified in the genus Butyricicoccus as Butyricicoccus desmolans comb. nov.

We investigated the viability and fatty acid synthetic activity of in vitro cultured Perkinsus marinus (Dermo) in lipid-free medium and estuarine water, and the infectivity of P. marinus maintained in artificial seawater (ASW). Viability and fatty acid synthetic activity in 7 d old P. marinus meronts maintained in lipid-free medium and estuarine water were tested. The infectivity of meronts incubated in ASW was examined by first incubating P. marinus meronts in ASW for 2, 3 or 7 d, and then inoculating viable ASW-incubated meronts into the shell cavity of individual oysters Crassostrea virginica. P. marinus infection prevalence and intensity in oysters were determined 9 wk post-inoculation. Heavy mortality occurred in meronts maintained in estuarine water, a drop from an initial value of 100% viable to 7.8 and 6.1% after 3 and 14 d incubation, respectively. Viability was 85 and 67% in meronts maintained in lipid-free medium for 3 and 24 d, respectively. Meronts kept in lipid-free medium for 14 d retained their ability to synthesize fatty acids. Viable meronts incubated in ASW remained infective for up to 7 d. The infection prevalences were 85, 48 and 100%, in the treatments inoculated with viable meronts that were incubated in ASW for 2, 3 and 7 d, respectively. Infection prevalence in the group inoculated with viable meronts immediately after they were transferred to ASW ranged from 61 to 85%. Our results suggest that in nature meronts can survive for at least 14 d outside the host. Viable meronts are not only infective, but are also able to replicate and retain their fatty acid synthetic ability for 7 d.

Rhodopsins are proteins that contain seven transmembrane domains with a chromophore retinal and that function as photoreceptors for light-energy conversion and light-signal transduction in a wide variety of organisms. Here we characterized a phylogenetically distinctive new rhodopsin from the thermophilic eubacterium Rubrobacter xylanophilus DSM 9941(T) that was isolated from thermally polluted water. Although R. xylanophilus rhodopsin (RxR) is from Actinobacteria, it is located between eukaryotic and archaeal rhodopsins in the phylogenetic tree. Escherichia coli cells expressing RxR showed a light-induced decrease in environmental pH and inhibition by a protonophore, indicating that it works as a light-driven outward proton pump. We characterized purified RxR spectroscopically, and showed that it has an absorption maximum at 541 nm and binds nearly 100% all-trans retinal. The pKa values for the protonated retinal Schiff base and its counterion were estimated to be 10.7 and 1.3, respectively. Time-resolved flash-photolysis experiments revealed the formation of a red-shifted intermediate. Of note, RxR showed an extremely high thermal stability in comparison with other proton pumping rhodopsins such as thermophilic rhodopsin TR (by 16-times) and bacteriorhodopsin from Halobacterium salinarum (HsBR, by 4-times).

The cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 is a modular enzyme comprised of a catalytic domain, a cellulose-binding domain and a cell wall-binding domain. Cellobiose-grown cells changed their adhesion ability to cellulose depending on the growth phase. On the other hand, carboxymethyl cellulose (CMC)-grown cells bound to cellulose regardless of their growth phase. The distribution of CBPA in the culture supernatant and cell fractions changed depending on the carbon source contained in the medium and growth phase. The cellobiose-grown cells harvested from the culture of the late stationary growth phase did not bind to cellulose, but their adhesion ability was recovered by treatment with recombinant CBPA. Moreover, cellobiose-grown cells harvested from the culture of an early exponential growth phase bound to cellulose, but their adhesion ability was inhibited by treatment with anti-CBPA antiserum. CBPA rapidly decreased the viscosity of CMC, indicating that CBPA was endoglucanase. The results obtained in this study indicate that CBPA plays an important role in the adhesion of E. cellulosolvens 5 cells to cellulose.

Eubacterium limosum ZL-II is an anaerobic bacterium with demethylated activity, which was isolated from human intestinal bacteria in our previous work. In this study, the flavonolignan constituents of Silybi Fructus were biotransformed by E. limosum(1) ZL-II, producing four new transformation products - demethylisosilybin B (T1), demethylisosilybin A (T2), demethylsilybin B (T3) and demethylsilybin A (T4), among which T1 and T2 were new compounds. Their chemical structures were identified by ESI-TOF/MS, (1)H NMR, (13)C NMR, HMBC and CD spectroscopic data. The bioassay results showed that the transformation products T1-T4 exhibited significant inhibitory activities on Alzheimer's amyloid-β 42 (Aβ42(2)) aggregation with IC50 values at 7.49 μM-10.46 μM, which were comparable with that of the positive control (epigallocatechin gallate, EGCG(3), at 9.01 μM) and much lower than those of their parent compounds (at not less than 145.10 μM). The method of biotransformation by E. limosum ZL-II explored a way to develop the new and active lead compounds in Alzheimer's disease from Silybi Fructus. However, the transformation products T1-T4 exhibited decreased inhibitory activities against human tumor cell lines comparing with their parent compounds.

Attempts were made to separate and characterize cellulose-binding proteins (CBPs) from both the culture supernatant and cell lysate of Eubacterium cellulosolvens 5. Once the CBPs were bound to Avicel cellulose, they were then effectively eluted with the solution containing 3.2 or 5% sodium dodecyl sulfate (SDS), but not eluted with the solution containing various kinds of carbohydrates and reagents. Namely, CBPs in both the culture supernatant and cell lysate of the bacterium bound tightly and strongly to cellulose. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted CBPs indicated that the CBPs contained the two major proteins having the molecular weights of approximately 160 and 84 kilodaltons (kDa) and one sub-major protein having a molecular weight of approximately 140 kDa. Zymogram analysis after the SDS-PAGE of the eluted CBPs showed that two proteins exhibited the highest levels of carboxymethyl cellulase (CMCase) activity corresponding to the molecular weights of approximately 160 and 90 kDa. A major protein having the molecular weight of approximately 160 kDa exhibited a distinct CMCase activity and was designated as CBPE1. Western immunoblot analysis indicated that the proteins prepared from 16 representative strains of rumen bacteria did not cross-react with rabbit antiserum raised against CBPE1. Thus, CBPE1 may be a unique CBP that plays an important role in the adhesion of the bacterium to cellulose.

Rhodopsins are proteins that contain seven transmembrane domains with a chromophore retinal and that function as photoreceptors for light-energy conversion and light-signal transduction in a wide variety of organisms. Here we characterized a phylogenetically distinctive new rhodopsin from the thermophilic eubacterium Rubrobacter xylanophilus DSM 9941T that was isolated from thermally polluted water. Although R. xylanophilus rhodopsin (RxR) is from Actinobacteria, it is located between eukaryotic and archaeal rhodopsins in the phylogenetic tree. Escherichia coli cells expressing RxR showed a light-induced decrease in environmental pH and inhibition by a protonophore, indicating that it works as a light-driven outward proton pump. We characterized purified RxR spectroscopically, and showed that it has an absorption maximum at 541 nm and binds nearly 100% all-trans retinal. The pKa values for the protonated retinal Schiff base and its counterion were estimated to be 10.7 and 1.3, respectively. Time-resolved flash-photolysis experiments revealed the formation of a red-shifted intermediate. Of note, RxR showed an extremely high thermal stability in comparison with other proton pumping rhodopsins such as thermophilic rhodopsin TR (by 16-times) and bacteriorhodopsin from Halobacterium salinarum (HsBR, by 4-times). PMID:28290523

Blood cell types of the sea lamprey, Petromyzon marinus, are described and identified and the lineage of mature circulating cells in peripheral blood is traced to blast cells in the hematopoietic fat body. The fat body appears to be the phylogenetic precursor of bone marrow in higher forms, since blood cells originate and begin maturation in this tissue. Experimental animals were injected first with a hematopoietic stimulant and then (at an experimentally determined time) with pertussis vaccine to release proliferated blood cells into peripheral blood. Peripheral blood for smears was collected by cardiac exsanguination; hematopoietic tissue was extirpated for imprints; and leucocyte preparations were made by a special technique. Blood cells of the sea lamprey are apparently products of at least four distinct blast cells, each of which has a 'one end' maturation process. Results of this investigation support the polyphyletic theory of blood cell formation.

Lentic populations of larval sea lampreys (Petromyzon marinus) are suspected of being a major source of recruitment to parasitic stocks in some areas of the Great Lakes, and methods are needed to estimate habitat and population sizes. A deepwater electroshocker has been used to quantitatively assess larval sea lamprey populations in deepwater areas, however a method has not been developed to efficiently identify the most promising locations to sample in this environment. A remote seabed classification device (RoxAnn™) was used to identify soft substrates in a lentic area where sea lamprey larvae have been found in Batchawana Bay (Ontario) in eastern Lake Superior, and related those substrate types to larval distribution and occurrence. Presence of larvae was significantly related to substrate type, distance from the stream mouth, and slope of the lake bottom. Remote seabed classification would be a useful tool in the Sea Lamprey Control Program to identify the most promising locations to conduct larval surveys in lentic areas.

The vertebrate gene family for neuropeptide Y (NPY) receptors expanded by duplication of the chromosome carrying the ancestral Y1-Y2-Y5 gene triplet. After loss of some duplicates, the ancestral jawed vertebrate had seven receptor subtypes forming the Y1 (including Y1, Y4, Y6, Y8), Y2 (including Y2, Y7) and Y5 (only Y5) subfamilies. Lampreys are considered to have experienced the same chromosome duplications as gnathostomes and should also be expected to have multiple receptor genes. However, previously only a Y4-like and a Y5 receptor have been cloned and characterized. Here we report the cloning and characterization of two additional receptors from the sea lamprey Petromyzon marinus. Sequence phylogeny alone could not with certainty assign their identity, but based on synteny comparisons of P. marinus and the Arctic lamprey, Lethenteron camtschaticum, with jawed vertebrates, the two receptors most likely are Y1 and Y2. Both receptors were expressed in human HEK293 cells and inositol phosphate assays were performed to determine the response to the three native lamprey peptides NPY, PYY and PMY. The three peptides have similar potencies in the nanomolar range for Y1. No obvious response to the three peptides was detected for Y2. Synteny analysis supports identification of the previously cloned receptor as Y4. No additional NPY receptor genes could be identified in the presently available lamprey genome assemblies. Thus, four NPY-family receptors have been identified in lampreys, orthologs of the same subtypes as in humans (Y1, Y2, Y4 and Y5), whereas many other vertebrate lineages have retained additional ancestral subtypes.

Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

This study investigated the length of avoidance response of migratory-stage sea lamprey Petromyzon marinus exposed continuously to conspecific damage-released alarm cues for varying lengths of time in laboratory stream channels. Ten replicate groups of P. marinus, separated by sex, were exposed to either deionized water control or to P. marinus extract for 0, 2 or 4 h continuously. Petromyzon marinus maintained their avoidance response to the conspecific damage-released alarm cue after continuous exposure to the alarm cue for 0 and 2 h but not 4 h. Beyond being one of the first studies in regards to sensory–olfactory adaptation–acclimation of fishes to alarm cues of any kind, these results have important implications for use of conspecific alarm cues in P. marinus control. For example, continuous application of conspecific alarm cue during the day, when P. marinus are inactive and hiding, may result in sensory adaptation to the odour by nightfall when they migrate upstream.

The eastern oyster, Crassostrea virginica (Gmelin), is both an important component of our estuaries and an important farmed food animal along the east and south coasts of the United States. Its populations have been significantly diminished in the wild due to decades of overfishing beginning in the 1890 s. Unfortunately, in 1950, a new disease in eastern oysters caused by the protistan agent, Perkinsus marinus, was identified. The disease, resulting from infection with this protozoan, leads to high mortality of both wild and cultured eastern oysters. Current restoration efforts are hampered by the disease, as is the aquaculture of this economically important food. The parasite infects hemocytes and causes hemolytic anemia and general degeneration of the tissues, leading to death. Ongoing research efforts are attempting to develop oysters resistant to the disease. Transport regulations exist in may states. Infection with P. marinus is listed as a reportable disease by the World Health Organization.

Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37 degrees C and 7.0, respectively. The K(m) for phloretin was 13 +/- 3 microM and the k(cat) was 10 +/- 2 s(-1). The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl(2) to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.

Resistant starch type 3 (RS3) is retrograded starch which is not digested by human starch degrading enzyme, and will thus undergo bacterial degradation in the colon. The main fermentation products are the Short Chain Fatty Acid (SCFA): acetate, propionate and butyrate. SCFA has significant benefit impact on the metabolism of the host. The objectives of this research were to study the SCFA profile produced by colonic butyrate producing bacteria grown in medium containing RS3. RS3 was made from sago or rice starch treated with amylase, pullulanase and the combination of amylase and pullulanase. Fermentation study was performed by using Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629, which has been identified as capable of degradation of starch residue and also regarded as beneficial bacteria. Experimental result revealed that enzyme hydrolysis of retrograded sago or rice starch was beneficial to RS formation. RS3 derived from sago contained higher RS (31-38%) than those derived from rice starch (21-26%). This study indicated that C. butyricum BCC B2571 produced acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1, when the medium was supplemented with RSSA at concentration 1%. In the medium containing similar substrate, E. rectale DSM 17629 produced acetate, propionate and butyrate at molar ratio of 1.7 : 1 : 1.2. High levels of acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1.1 was also produced by E. rectale DSM 17629 in medium supplemented with RSSP at concentration 1%. The results showed that both bacteria responded differently to the RS3 supplementation. Such result provided insight into the possibility of designing RS3 as prebiotic with featured regarding SCFA released in the human colon with potential health implication.

The protozoan parasite Perkinsus marinus is the etiological agent of "dermo disease". This pathogen is considered by the World Organization for Animal Health (OIE) as reportable due to the high mortalities that it produces in the eastern oyster Crassostrea virginica in the US. In 2006, this parasite was detected in the pleasure oyster Crassostrea corteziensis in Nayarit on the Pacific coast of Mexico, indicating a new host and an extension of its known distribution. Epizootiological data of P. marinus in the pleasure oyster are unknown. With the objective of determining the prevalence and intensity in relation with temperature and salinity throughout time, as well as for studying interactions of host size and sex with the parasite, a monthly sampling was carried out in two aquaculture sites of Nayarit from 2007 to 2014. A total of 7700 oysters were analyzed. In both localities, prevalence was low in winter (<6%) when temperature and salinity fluctuated around 24°C and 33, respectively; and the highest prevalence values occurred during summer (37%) when temperature and salinity were around 30°C and 20, respectively. Infection intensity increased in summer, but severe cases remained on average <10%. Larger oysters showed the highest prevalence and intensity, and higher prevalence were generally observed in females. No unusual mortalities directly related with P. marinus were observed.

Studies were made on the composition of the growth medium to increase the cell concentration in a cell-recycled continuous culture (Eubacterium limosum KIST612) with carbon monoxide as a sole energy source using phosphate-buffered basal medium (PBBM) and modified PBBM. One of major limiting factors in PBBM might be nitrogen during the high cell density culture. This limitation could be overcome by increasing of inorganic nitrogen or yeast extract concentration in the medium. Anaerobic digester fluid, which could replace the organic nitrogen in PBBM, was used to develop an industrial grade medium for conversion of CO to multi-carbon compound.

Phylogenetic analysis was used to compare 16S rRNA sequences from 19 cultured human gut strains of Roseburia and Eubacterium rectale with 356 related sequences derived from clone libraries. The cultured strains were found to represent five of the six phylotypes identified. A new oligonucleotide probe, Rrec584, and the previous group probe Rint623, when used in conjunction with a new helper oligonucleotide, each recognized an average of 7% of bacteria detected by the eubacterial probe Eub338 in feces from 10 healthy volunteers. Most of the diversity within this important group of butyrate-producing gut bacteria can apparently be retrieved through cultivation.

Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for “Dermo” disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1–2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA0) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA0 mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA0 mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA0 mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents. PMID:24498105

Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for "Dermo" disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1-2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA(0)) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA(0) mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA(0) mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA(0) mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents.

Copepods are small crustaceans capable to survive in various aquatic environments. Their responses to changes in different external factors such as salinity and temperature can be observed at different integration levels from copepod genes to copepod communities. Until now, no thorough observation of the temperature or salinity effect stresses on copepods has been done by optical microscopy. In this study, we used autofluorescence to visualize these effects on the morphology of the calanoid copepod Pseudodiaptomus marinus maintained during several generations in the laboratory at favorable and stable conditions of salinity (30 psu) and temperature (18°C). Four different stress experiments were conducted: at a sharp decrease in temperature (18 to 4°C), a moderate decrease in salinity (from 30 to 15 psu), a major decrease in salinity (from 30 to 0 psu), and finally a combined stress with a decrease in both temperature and salinity (from 18°C and 30 psu to 4°C and 0 psu). After these stresses, images acquired by confocal laser scanning microscopy (CLSM) revealed changes in copepod cuticle and muscle structure. Low salinity and/or temperature stresses affected both the detection of fluorescence emitted by muscle sarcomeres and the distance between them. In the remaining paper we will use the term sarcomeres to describe the elements located within sarcomeres and emitted autofluorescence signals. Quantitative study showed an increase in the average distance between two consecutive sarcomeres from 2.06 +/- 0.11 μm to 2.44 +/- 0.42 μm and 2.88 +/- 0.45μm after the exposure to major haline stress (18°C, 0 psu) and the combined stress (4°C, 0 psu), respectively. These stresses also caused cuticle cracks which often occurred at the same location, suggesting the cuticle as a sensitive area for osmoregulation. Our results suggest the use of cuticular and muscle autofluorescence as new biomarkers of stress detectable in formalin-preserved P. marinus individuals. Our

In the past decade, there have been increasing concerns over the effects of pharmaceutical compounds in the aquatic environment, however very little is known about the effects of antidepressants such as the selective serotonin re-uptake inhibitors (SSRIs). Many biological functions within invertebrates are under the control of serotonin, such as reproduction, metabolism, moulting and behaviour. The effects of serotonin and fluoxetine have recently been shown to alter the behaviour of the marine amphipod, Echinogammarus marinus (Leach, 1815). The purpose of this study was to observe behavioural and transcriptional modifications in this crustacean exposed to the two most prescribed SSRIs (fluoxetine and sertraline) and to develop biomarkers of neurological endocrine disruption. The animals were exposed to both drugs at environmentally relevant concentrations from 0.001 to 1μg/L during short-term (1h and 1day) and medium-term (8 days) experiments. The movement of the amphipods was tracked using the behavioural analysis software during 12min alternating dark/light conditions. The behavioural analysis revealed a significant effect on velocity which was observed after 1h exposure to sertraline at 0.01μg/L and after 1 day exposure to fluoxetine as low as 0.001μg/L. The most predominant effect of drugs on velocity was recorded after 1 day exposure for the 0.1 and 0.01μg/L concentrations of fluoxetine and sertraline, respectively. Subsequently, the expression (in this article gene expression is taken to represent only transcription, although it is acknowledged that gene expression can also be regulated at translation, mRNA and protein stability levels) of several E. marinus neurological genes, potentially involved in the serotonin metabolic pathway or behaviour regulation, were analysed in animals exposed to various SSRIs concentrations using RT-qPCR. The expression of a tryptophan hydroxylase (Ph), a neurocan core protein (Neuc), a Rhodopsin (Rhod1) and an Arrestin

Two similar gram-positive rods were isolated from 10(-6) dilutions of ruminal fluid from a sheep receiving a mixed grass hay/concentrate diet, using a medium containing pancreatic casein hydrolysate as sole source of carbon and energy. The isolates did not ferment sugars, but grew on pyruvate or trypticase, forming caproate as the main fermentation product and valerate to a lesser extent. Acetate and propionate were utilized. One of these strains, I-6T, was selected for further study. Strain I-6T was a non-motile coccal rod, 1.2 x 0.4 microm, with a gram-positive cell wall ultrastructure and a G + C content of 56.8 mol%. No spores were visible, and strain I-6T did not survive heating at 80 degrees C for 10 min. Its rate of NH3 production was 375 nmol (mg protein)(-1) min(-1), placing it in the 'ammonia-hyperproducing' (or HAP) group of ruminal bacteria. 16S rDNA sequence analysis (1296 bases) indicated that it represents a novel species within the 'low-G + C' gram-positive group, for which the name Eubacterium pyruvativorans sp. nov. is proposed. Among cultivated bacteria, strain I-6T was most closely related (89% identity) to other asaccharolytic Eubacterium isolates from the mouth and the rumen. It was 98% identical to uncultured bacterial sequences amplified by others from ruminal digesta.

Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 degrees C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source.

Restoration of oyster reef habitat in the Inland Bays of Delaware was accompanied by an effort to detect and determine relative abundance of the bivalve pathogens Perkinsus marinus, Haplosporidium nelsoni, and QPX. Both the oyster Crassostrea virginica and the clam Mercenaria mercenaria were sampled from the bays. In addition, oysters were deployed at eight sites around the bays as sentinels for the three parasites. Perkinsus marinus prevalence was measured with a real-time, quantitative polymerase chain reaction (PCR) methodology that enabled high-throughput detection of as few as 31 copies of the ribosomal non-transcribed spacer region in 500 ng oyster DNA. The other pathogens were assayed using PCR with species-specific primers. Perkinsus marinus was identified in Indian River Bay at moderate prevalence ( approximately 40%) in both an artificial reef and a wild oyster population whereas sentinel oysters were PCR-negative after 3-months exposure during summer and early fall. Haplosporidium nelsoni was restricted to one oyster deployed in Little Assawoman Bay. QPX and P. marinus were not detected among wild clams. While oysters in these bays have historically been under the greatest threat by MSX, it is apparent that P. marinus currently poses a greater threat to recovery of oyster aquaculture in Delaware's Inland Bays.

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called “CLARITY” was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

We used lengths and weights of 2367 live parasitic-phase sea lampreys (Petromyzon marinus) collected from Lake Huron, 1984–1990, to calculate their mean size at half-month intervals. Growth in weight was linear during June through September; increments averaged 11.1 g per half month. Growth increased sharply in October to several times the summer rate. We speculate that the increase in growth in October is explained partly by water temperature and partly by an increase in appetite related to the onset of gonadal development. The greater compression of biomass accumulation in autumn than has been previously demonstrated better explains the autumn pulse of sea lamprey induced host mortality. Based on the seasonal pattern of growth and on recaptures of marked sea lampreys, we conclude that landlocked individuals grow to adult size and mature in one parasitic growth year. Regressions of weight (grams) on total length (millimetres) differed significantly among months, and the season of collection must be considered in predicting weight from length.

Release of males sterilized by injection with bisazir is an important experimental technique in management of sea lamprey (Petromyzon marinus), an invasive, nuisance species in the Laurentian Great Lakes. Sea lampreys are semelparous and sterilization can theoretically eliminate a male's reproductive capacity and, if the ability to obtain mates is not affected, waste the sex products of females spawning with him. It has been demonstrated that spermiating males release a sex pheromone that attracts ovulating females. We demonstrated that sterilized, spermiating males also released the pheromone and attracted ovulating females. In a two-choice maze, ovulating females increased searching behavior and spent more time in the side of the maze containing chemical stimuli from sterilized, spermiating males. This attraction response was also observed in spawning stream experiments. Also, electro-olfactograms showed that female olfactory organs were equally sensitive to chemical stimuli from sterilized and nonsterilized, spermiating males. Finally, fast atom bombardment mass spectrometry showed that extracts from water conditioned with sterilized and nonsterilized, spermiating males contained the same pheromonal molecule at similar levels. We concluded that injection of bisazir did not affect the efficacy of sex pheromone in sterilized males.

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1–4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N′ domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N′ domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes. PMID:22223643

The role of the C24 sulphate in the mating pheromone component, 7α,12α,24-trihydroxy-5α-cholan-3-one 24-sulphate (3kPZS), to specifically induce upstream movement in ovulated female sea lampreys Petromyzon marinus was investigated. 7α,12α-dihydroxy-5α-cholan-3-one 24-oic acid (3kACA), a structurally similar bile acid released by spermiated males, but lacking the C24 sulphate ester, was tested in bioassays at concentrations between 10−11 and 10−14 molar (M). 3kACA did not induce upstream movement in females or additional reproductive behaviours. In contrast, spermiated male washings induced upstream movement, prolonged retention on a nest and induced an array of nesting behaviours. Differential extraction and elution by solid-phase extraction resins showed that components other than 3kPZS + 3kACA are necessary to retain females on nests and induce nest cleaning behaviours. All pheromone components, including components in addition to 3kPZS + 3kACA that retain females and induce nest cleaning behaviours were released from the anterior region of the males, as had been reported for 3kPZS. It is concluded that the sea lamprey male mating pheromone has multiple functions and is composed of multiple components.

Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.

Application of chemical cues to manipulate adult sea lamprey (Petromyzon marinus) behavior is among the options considered for new sea lamprey control techniques in the Laurentian Great Lakes. A male mating pheromone component, 7a,12a,24-trihydroxy-3-one-5a-cholan-24-sulfate (3kPZS), lures ovulated female sea lamprey upstream into baited traps in experimental contexts with no odorant competition. A critical knowledge gap is whether this single pheromone component influences adult sea lamprey behavior in management contexts containing free-ranging sea lampreys. A solution of 3kPZS to reach a final in-stream concentration of 10-12 mol·L-1 was applied to eight Michigan streams at existing sea lamprey traps over 3 years, and catch rates were compared between paired 3kPZS-baited and unbaited traps. 3kPZS-baited traps captured significantly more sexually immature and mature sea lampreys, and overall yearly trapping efficiency within a stream averaged 10% higher during years when 3kPZS was applied. Video analysis of a trap funnel showed that the likelihood of sea lamprey trap entry after trap encounter was higher when the trap was 3kPZS baited. Our approach serves as a model for the development of similar control tools for sea lamprey and other aquatic invaders.

The Geodermatophilaceae are unique model systems to study the ability to thrive on or within stones and their proteogenomes (referring to the whole protein arsenal encoded by the genome) could provide important insight into their adaptation mechanisms. Here we report the detailed comparative genome analysis of Blastococcus saxobsidens (Bs), Modestobacter marinus (Mm) and Geodermatophilus obscurus (Go) isolated respectively from the interior and the surface of calcarenite stones and from desert sandy soils. The genome-scale analysis of Bs, Mm and Go illustrates how adaptation to these niches can be achieved through various strategies including ‘molecular tinkering/opportunism' as shown by the high proportion of lost, duplicated or horizontally transferred genes and ORFans. Using high-throughput discovery proteomics, the three proteomes under unstressed conditions were analyzed, highlighting the most abundant biomarkers and the main protein factors. Proteomic data corroborated previously demonstrated stone-related ecological distribution. For instance, these data showed starvation-inducible, biofilm-related and DNA-protection proteins as signatures of the microbes associated with the interior, surface and outside of stones, respectively. PMID:26125681

Lake trout (Salvelinus namaycush) was exterminated in Lake Michigan by the mid-1950s as a result of the combined effects of an intensive fishery and predation by the sea lamprey (Petromyzon marinus). The widespread application of lampricide in tributary streams had greatly reduced the abundance of lampreys by the early 1960s, and a program to restore self-sustaining populations of lake trout through stocking of yearlings and fingerlings was initiated in 1965. Although the hatchery-reared fish spawned widely in Lake Michigan each year after 1970, no progeny were observed except in an isolated area in Grand Traverse Bay. During 1971–78, sea lamprey abundance was generally greater in Wisconsin than in other parts of the lake. However, the rate of occurrence of sea lamprey wounds on lake trout dropped dramatically there in 1978 after the Peshtigo River, a tributary to Green Bay, was treated with lampricide. Application of Lake Michigan wounding rates to a regression model relating mortality to lamprey wounding developed from Lake Superior data, yielded lamprey-induced mortality estimates in 1977 of 5% in Michigan plus Indiana (combined) and 31% in Wisconsin; corresponding estimates for 1978 were 5 and 15%.Key words: lake trout, sea lamprey predation, abundance, Lake Michigan

We examined the ontogenetic dynamics of mercury accumulation in sea lamprey (Petromyzon marinus) from the Connecticut River, USA. Mercury concentrations in eggs (mean 84 ng??g-1 wet weight) were lowest of all life stages and correlated to concentrations in females. There was a higher rate of maternal transfer of mercury to eggs compared with teleosts. Ammocoetes had high mercury concentrations for their trophic level (e.g., mean of age-4 ammocoetes 492 ng??g-1 wet weight). A further investigation of four streams showed that ammocoetes reflected the level of contamination in their nursery streams. Concentrations of mercury decreased during metamorphosis from ammocoete to adult. Mercury concentrations in adults ranged from 83 to 942 ng??g-1 wet weight and, unlike teleosts, showed no relation to sex, length, or weight. We provide evidence from stable isotope analyses that this high variability is due to feeding ecology. There are fundamental differences in mercury accumulation between sea lamprey and teleosts. ?? 2006 NRC Canada.

The control of invasive sea lampreys (Petromyzon marinus) presents large scale management challenges in the Laurentian Great Lakes. No modeling approach has been developed that describes spatial dynamics of lamprey populations. We developed and validated a spatial and age-structured model and applied it to a sea lamprey population in a large river in the Great Lakes basin. We considered 75 discrete spatial areas, included a stock-recruitment function, spatial recruitment patterns, natural mortality, chemical treatment mortality, and larval metamorphosis. Recruitment was variable, and an upstream shift in recruitment location was observed over time. From 1993–2011 recruitment, larval abundance, and the abundance of metamorphosing individuals decreased by 80, 84, and 86%, respectively. The model successfully identified areas of high larval abundance and showed that areas of low larval density contribute significantly to the population. Estimated treatment mortality was less than expected but had a large population-level impact. The results and general approach of this work have applications for sea lamprey control throughout the Great Lakes and for the restoration and conservation of native lamprey species globally.

The Geodermatophilaceae are unique model systems to study the ability to thrive on or within stones and their proteogenomes (referring to the whole protein arsenal encoded by the genome) could provide important insight into their adaptation mechanisms. Here we report the detailed comparative genome analysis of Blastococcus saxobsidens (Bs), Modestobacter marinus (Mm) and Geodermatophilus obscurus (Go) isolated respectively from the interior and the surface of calcarenite stones and from desert sandy soils. The genome-scale analysis of Bs, Mm and Go illustrates how adaptation to these niches can be achieved through various strategies including 'molecular tinkering/opportunism' as shown by the high proportion of lost, duplicated or horizontally transferred genes and ORFans. Using high-throughput discovery proteomics, the three proteomes under unstressed conditions were analyzed, highlighting the most abundant biomarkers and the main protein factors. Proteomic data corroborated previously demonstrated stone-related ecological distribution. For instance, these data showed starvation-inducible, biofilm-related and DNA-protection proteins as signatures of the microbes associated with the interior, surface and outside of stones, respectively.

Four electrical factors that are used in pulsed DC electrofishing for larval sea lampreys (Petromyzon marinus) were evaluated in two laboratory studies to determine the optimal values to induce larval emergence over a range of water temperatures and conductivities. Burrowed larvae were exposed to combinations of pulsed DC electrical factors including five pulse frequencies, three pulse patterns, and two levels of duty cycle over a range of seven voltage gradients in two separate studies conducted at water temperatures of 10, 15, and 20°C and water conductivities of 25, 200, and 900 μS/cm. A four-way analysis of variance was used to determine significant (α = 0.05) influences of each electrical factor on larval emergence. Multiple comparison tests with Bonferroni adjustments were used to determine which values of each factor resulted in significantly higher emergence at each temperature and conductivity. Voltage gradient and pulse frequency significantly affected emergence according to the ANOVA model at each temperature and conductivity tested. Duty cycle and pulse pattern generally did not significantly influence the model. Findings suggest that a setting of 2.0 V/cm, 3 pulses/sec, 10% duty, and 2:2 pulse pattern seems the most promising in waters of medium conductivity and across a variety of temperatures. This information provides a basis for understanding larval response to pulsed DC electrofishing gear factors and identifies electrofisher settings that show promise to increase the efficiency of the gear during assessments for burrowed sea lamprey larvae.

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called "CLARITY" was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

We determined the polychlorinated biphenyl (PCB) concentrations of 40 male and 40 female adult sea lampreys Petromyzon marinus captured in the Cheboygan River, a tributary to Lake Huron, during May 2011. In addition, we performed a laboratory experiment using passive integrated transponder tags to determine whether male adult sea lampreys were more active than female adult sea lampreys. Sex had a significant effect on PCB concentration, and PCB concentration at a given level of sea lamprey condition was approximately 25 % greater in males than in females. Adjusting for the difference in condition between the sexes, males averaged a 17 % greater PCB concentration compared with females. Results from the laboratory experiment indicated that males were significantly more active than females. The observed sex difference in PCB concentrations was not due to female sea lampreys releasing eggs at spawning because the sea lamprey is semelparous, and we caught the sea lampreys before spawning. Rather, we attributed the sex difference in PCB concentrations to a greater rate of energy expenditure in males compared with females. We proposed that this greater rate of energy expenditure was likely due to greater activity. Our laboratory experiment results supported this hypothesis. A greater resting metabolic rate may also have contributed to a greater rate of energy expenditure. Our findings should eventually be applicable toward improving control of sea lamprey, a pest responsible for considerable damage to fisheries in lakes where it is not native.

Metamorphosed sea lampreys (Petromyzon marinus) were collected and marked at two points in their life cycle. Recently metamorphosed juveniles were collected from streams, marked with coded wire tags, and returned to migrate to the Great Lakes. Juveniles already in the lakes and feeding on teleost hosts were obtained from incidental catches by sport or commercial fisheries. Sea lampreys in the Great Lakes spend only 1 feeding year as parasites, and marked animals were recaptured during the spawning runs. For one marked group in each of four parasitic cohorts (feeding years 1991 to 1994) and two marked groups in each of three cohorts (feeding years 1998 to 2000) we recovered from 1.1 to 10.2 percent of marked animals. The number of metamorphosed animals present in autumn before migration to Lake Huron was estimated for five cohorts, with estimates ranging from 639 to 803 thousand. The number of feeding, parasitic animals present in Lake Huron in mid summer was estimated for five cohorts, with estimates ranging from 515,000 to 2,342,000. The larger estimates later in the parasitic year suggested that animals collected and marked from sport or commercial fisheries did not survive at the same rate as unmarked animals. It is recommended that only estimates from recaptures of animals marked in the streams before migration be used until it can be established why survival of juveniles obtained from sport or commercial fisheries might be affected.

Criteria for the classification of marks inflicted by sea lamprey (Petromyzon marinus) into nine categories were developed from laboratory studies in an attempt to refine the classification system used in field assessment work. These criteria were based on characteristics of the attachment site that could be identified under field conditions by unaided visual means and by touching the attachment site. Healing of these marks was somewhat variable and was influenced by the size of lamprey, duration of attachment, severity of the wound at lamprey detachment, season and water temperature, and by other less obvious factors. Even under laboratory conditions staging of some wounds was difficult, especially at low water temperatures. If these criteria are to be used effectively and with precision in the field, close examination of individual fish may be required. If the feeding and density of specific year-classes of sea lampreys are to be accurately assessed on an annual basis, close attention to the wound size (as it reflects the size of the lamprey's oral disc) and character of wounds on fish will be required as well as consideration of the season of the year in which they are observed.Key words: sea lamprey, attack marks, lake trout, Great Lakes

Stream-finding behavior of adult sea lamprey (Petromyzon marinus), an anadromous fish that relies on pheromones to locate spawning streams, was documented in the vicinity of an important spawning river in the Great Lakes. Untreated and anosmic migrating sea lampreys were implanted with acoustic transmitters and then released outside the Ocqueoc River. Lampreys swam only at night and then actively. When outside of the river plume, lampreys pursued relatively straight bearings parallel to the shoreline while making frequent vertical excursions. In contrast, when within the plume, lampreys made large turns and exhibited a weak bias towards the river mouth, which one-third of them entered. The behavior of anosmic lampreys resembled that of untreated lampreys outside of the plume, except they pursued a more northerly compass bearing. To locate streams, sea lampreys appear to employ a three-phase odor-mediated strategy that involves an initial search along shorelines while casting vertically, followed by river-water-induced turning that brings them close to the river's mouth, which they then enter using rheotaxis. This novel strategy differs from that of salmonids and appears to offer this poor swimmer adaptive flexibility and suggests ways that pheromonal odors might be used to manage this invasive species.

Synchronization of male and female locomotor rhythmicity can play a vital role in ensuring reproductive success. Several physiological and environmental factors alter these locomotor rhythms. As sea lamprey, Petromyzon marinus, progress through their life cycle, their locomotor activity rhythm changes multiple times. The goal of this study was to elucidate the activity patterns of adult female sea lamprey during the sexual maturation process and discern the interactions of these patterns with exposure to male pheromones. During these stages, preovulated and ovulated adult females are exposed to sex pheromone compounds, which are released by spermiated males and attract ovulated females to the nest for spawning. The locomotor behavior of adult females was monitored in a natural stream with a passive integrated tag responder system as they matured, and they were exposed to a sex pheromone treatment (spermiated male washings) or a control (prespermiated male washings). Results showed that, dependent on the hour of day, male sex pheromone compounds reduce total activity (p < 0.05) and cause increases in activity during several daytime hours in preovulated and ovulated females. These results are one of the first examples of how sex pheromones modulate a locomotor rhythm in a vertebrate, and they suggest that the interaction between maturity stage and sex pheromone exposure contributes to the differential locomotor rhythms found in adult female sea lamprey. This phenomenon may contribute to the reproductive synchrony of mature adults, thus increasing reproductive success in this species.

Non-physical stimuli can deter or guide fish without affecting water flow or navigation and therefore have been investigated to improve fish passage at anthropogenic barriers and to control movement of invasive fish. Upstream fish migration can be blocked or guided without physical structure by electrifying the water, but directional downstream fish guidance with electricity has received little attention. We tested two non-uniform pulsed direct current electric systems, each having different electrode orientations (vertical versus horizontal), to determine their ability to guide out-migrating juvenile sea lamprey (Petromyzon marinus) and rainbow trout (Oncorhynchus mykiss). Both systems guided significantly more juvenile sea lamprey to a specific location in our experimental raceway when activated than when deactivated, but guidance efficiency decreased at the highest water velocities tested. At the electric field setting that effectively guided sea lamprey, rainbow trout were guided by the vertical electrode system, but most were blocked by the horizontal electrode system. Additional research should characterize the response of other species to non-uniform fields of pulsed DC and develop electrode configurations that guide fish over a range of water velocity.

Prediction of how host mortality responds to efforts to control sea lampreys (Petromyzon marinus) is central to the integrated management strategy for sea lamprey (IMSL) in the Great Lakes. A parasite-host submodel is used as part of this strategy, and this includes a type-2 multi-species functional response, a developmental response, but no numerical response. General patterns of host species and size selection are consistent with the model assumptions, but some observations appear to diverge. For example, some patterns in sea lamprey marking on hosts suggest increases in selectivity for less preferred hosts and lower host survival when preferred hosts are scarce. Nevertheless, many of the IMSL assumptions may be adequate under conditions targeted by fish community objectives. Of great concern is the possibility that the survival of young parasites (parasitic-phase sea lampreys) varies substantially among lakes or over time. Joint analysis of abundance estimates for parasites being produced in streams and returning spawners could address this. Data on sea lamprey marks is a critical source of information on sea lamprey activity and potential effects. Theory connecting observed marks to sea lamprey feeding activity and host mortality is reviewed. Uncertainties regarding healing and attachment times, the probability of hosts surviving attacks, and problems in consistent classification of marks have led to widely divergent estimates of damages caused by sea lamprey. Laboratory and field studies are recommended to provide a firmer linkage between host blood loss, host mortality, and observed marks on surviving hosts, so as to improve estimates of damage.

In this study we developed an SDS-PAGE protocol which for the first time separates effectively all myosin heavy chain (MHC) isoforms expected to be expressed in iliofibularis (IF), pyriformis (PYR), cruralis (CRU) and sartorius (SAR) muscles of the toad Bufo marinus on the basis of previously reported fibre type composition. The main feature of the method is the use of alanine instead of glycine both in the separating gel and in the running buffer. The correlation between the MHC isoform composition of IF, SAR and PYR muscles determined in this study and the previously reported fibre type composition of IF and SAR muscles in the toad and of PYR muscle in the frog was used to tentatively identify the MHC isoforms expressed by twitch fibre types 1, 2 and 3 and by tonic fibres. The alanine-SDS electrophoretic method was employed to examine changes in the MHC composition of IF, PYR, CRU and SAR muscles with the ontogenetic growth of the toad from post-natal life (body weight < 1 g) to late adulthood (body weight 200-450 g). The developmental changes in the MHC isoform composition of the toad IF muscle observed in this study are in very good agreement with those in the fibre type composition of the developing IF muscle reported in the literature.

Most work examining muscle function during anuran locomotion has focused largely on the roles of major hind limb extensors during jumping and swimming. Nevertheless, the recovery phase of anuran locomotion likely plays a critical role in locomotor performance, especially in the aquatic environment, where flexing limbs can increase drag on the swimming animal. In this study, I use kinematic and electromyographic analyses to explore the roles of four anatomical flexor muscles in the hind limb of Bufo marinus during swimming: m. iliacus externus, a hip flexor; mm. iliofibularis and semitendinosus, knee flexors; and m. tibialis anticus longus, an ankle flexor. Two general questions are addressed: (1) What role, if any, do these flexors play during limb extension? and (2) How do limb flexors control limb flexion? Musculus iliacus externus exhibits a large burst of EMG activity early in limb extension and shows low levels of activity during recovery. Both m. iliofibularis and m. semitendinosus are biphasically active, with relatively short but intense bursts during limb extension followed by longer and typically weaker secondary bursts during recovery. Musculus tibialis anticus longus becomes active mid way through recovery and remains active through the start of extension in the next stroke. In conclusion, flexors at all three joints exhibit some activity during limb extension, indicating that they play a role in mediating limb movements during propulsion. Further, recovery is controlled by a complex pattern of flexor activation timing, but muscle intensities are generally lower, suggesting relatively low force requirements during this phase of swimming.

Sea lamprey (Petromyzon marinus) avoid damage-released and predator chemosensory cues at night, but their response to these cues during the day is unknown. Here, we explored (i) whether sea lamprey avoid these cues during the day and (ii) the effect of water temperature on the avoidance of chemosensory alarm cues in two diurnal laboratory experiments. We hypothesized that daytime activity would be temperature-dependent and that only sea lamprey vulnerable to predation (i.e., not hiding) would behaviourally respond to chemosensory alarm cues. Ten groups of ten sea lamprey were exposed to one of a variety of potential chemosensory cues. The experiments were conducted over a range of temperatures to quantify the effect of temperature on avoidance behaviour. Consistent with our hypothesis, a higher proportion of animals were active during daytime as water temperature increased. Moving sea lamprey showed an avoidance response to 2-phenylethylamine (a compound found in mammalian urine) and human saliva once water temperatures had risen to mean (±SD) = 13.7 (±1.4) °C. Resting and hiding sea lamprey did not show an avoidance response to any of the experimental stimuli.

Understanding the environmental parameters that constrain the distribution of a species at its latitudinal extremes is critical for predicting how ecosystems react to climate change. Our first aim was to predict the variation in the amphipod populations of Echinogammarus marinus from the southernmost limit of its distribution under global warming scenarios. Our second aim was to test whether sex-ratio fluctuations - a mechanism frequently displayed by amphipods - respond to the variations in populations under altered climate conditions. To achieve these aims, scenarios were run with a validated model of E. marinus populations. Simulations were divided into: phase I - simulation of the effect of climate change on amphipod populations, and phase II - simulation of the effect of climate change on populations with male and female proportions. In both phases, temperature (T), salinity (S) and temperature and salinity (T-S) were tested. Results showed that E. marinus populations are highly sensitive to increases in temperature (>2 °C), which has adverse effects on amphipod recruitment and growth. Results from the climate change scenarios coupled with the sex-ratio fluctuations depended largely on the degree of female bias within population. Temperature increase of 2 °C had less impact on female-biased populations, particularly when conjugated with increases in salinity. Male-biased populations were highly sensitive to any variation in temperature and/or salinity; these populations exhibited a long-term decline in density. Simulations in which temperature increased more than 4 °C led to a continuous decline in the E. marinus population. According to this work, E. marinus populations at their southernmost limit are vulnerable to global warming. We anticipate that in Europe, temperature increases of 2 °C will incite a withdrawal of the population of 5°N from the amphipod species located at southernmost geographical borders. This effect is discussed in relation to the

The captive breeding program for the endangered Puerto Rican crested toad (Peltophryne [Bufo] lemur) has been hampered by an undiagnosed condition called "Brown Skin Disease" (BSD). Toads develop widespread skin darkening, skin thickening and abnormal shedding and eventually succumb to a chronic loss of viability. This project evaluated the marine toad (Bufo marinus) as a model for the PRCT, examining vitamin A deficiency as a potential cause of BSD. Wild caught marine toads had significantly higher liver vitamin A concentrations (61.89 ± 63.49 µg/g) than captive born marine toads (0.58 ± 0.59 µg/g); P<0.001). A significant difference in serum vitamin A concentration was found between the captive and wild caught toads (P=0.013) and between the low vitamin A-fed and wild caught toads (P=0.004), when controlling for liver vitamin A concentrations. After captive toads were treated with topical and/or oral vitamin A, their hepatic vitamin A concentrations were similar to those of the wild toads, averaging 48.41 ± 37.03 µg/g. However, plasma vitamin A concentrations pre- and post-vitamin A supplementation did not differ statistically. We concluded that plasma vitamin A concentrations do not provide a linear indication of liver/body vitamin A status, and that both topical and oral supplementation with an oil-based vitamin A formulation can increase liver stores in amphibians. No evidence of BSD or other signs of deficiency were noted in the marine toads, although this feeding trial was relatively short (127 days). To date, clinical, pathological and research findings do not support vitamin A deficiency as a primary factor underlying BSD.

Sea lampreys (Petromyzon marinus) disturb the substratum during nest construction and alter the physical habitat, potentially affecting other stream organisms. We quantified differences in depth, velocity, fine-sediment coverage, embeddedness, intragravel permeability and benthic invertebrate assemblages (density and diversity) among nest mounds, nest pits and undisturbed reference locations over a 4-month period after June spawning. In 2010 and 2011, immediate and persistent effects of nest construction were assessed in summer (July) and in autumn (late September to early October), respectively. Randomly selected nests were sampled annually (25 each in summer and autumn). Nest construction increased stream-bed complexity by creating and juxtaposing shallow, swift, rocky habitat patches with deep, slow, sandy habitat patches. Mounds had a 50–143% less cover of fine sediment, and a 30–62% reduction in embeddedness, compared to pits and reference locations. These physical changes persisted into the autumn (almost 4 months). Five insect families contributed 74% of the benthic invertebrate abundance: Chironomidae (27%), Hydropsychidae (26%), Heptageniidae (8%), Philopotamidae (7%) and Ephemerellidae (6%). Densities of Hydropsychidae, Philopotamidae and Heptageniidae were up to 10 times greater in mounds than in pits and adjacent reference habitat. In summer, mounds had twice the density of Chironomidae than did pits, and 1.5 times more than reference habitats, but densities were similar among the habitats in autumn. These results suggest that spawning sea lampreys are ecosystem engineers. The physical disturbance caused by nest-building activity was significant and persistent, increasing habitat heterogeneity and favouring pollution-sensitive benthic invertebrates and, possibly, drift-feeding fish.

Microbial pathogens succeed in acquiring essential metals such as iron and manganese despite their limited availability because of the host's immune response. The eukaryotic natural resistance-associated macrophage proteins mediate uptake of divalent metals and, during infection, may compete directly for metal acquisition with the pathogens' transporters. In this study, we characterize the Nramp gene family of Perkinsus marinus, an intracellular parasite of the eastern oyster, and through yeast complementation, we demonstrate for the first time for a protozoan parasite that Nramp imports environmental Fe. Three PmNramp isogenes differ in their exon-intron structures and encode transcripts that display a trans splicing leader at the 5' end. The protein sequences share conserved properties predicted for the Nramp/Solute carrier 11 (Slc11) family, such as 12-transmembrane segment (TMS) topology (N- and C-termini cytoplasmic) and preferential conservation of four TMS predicted to form a pseudosymmetric proton/metal symport pathway. Yeast fet3fet4 mutant complementation assays showed iron transport activity for PmNramp1 and a fusion chimera of the PmNramp3 hydrophobic core and PmNramp1 N- and C-termini. PmNramp1 site-directed mutagenesis demonstrated that Slc11 invariant and predicted pseudosymmetric motifs (TMS1 Asp-Pro-Gly and TMS6 Met-Pro-His) are key for transport function. PmNramp1 TMS1 mutants D76E, G78A, and D76E/G78A prevented membrane protein expression, while TMS6 M250A, H252Y, and M250A/H252Y specifically abrogated Fe uptake; the TMS6 H252Y mutation also correlates with divergence from Nramp specificity for divalent metals.

Melanin is a pigment widely occurring in animals, plants, fungi and algae. It does not only colour skin, hair and eyes but serves mainly as photoprotectant and prevents overload with minerals induced by inflammations, infections and degenerative diseases. Therefore, the mechanisms underlying melanisation gained increasing interest in the field of biomedical research and clinic. So far, the processes of melanogenesis are only partly analysed, nearly nothing is known on a putative switch between melanins of different types. Here we offer a model organism to study these mechanisms as part of a naturally cycling change of transparency of the retinal shielding pigment. A marine midge, Clunio marinus, living in coastal regions, underlies a complex timing of its development by solar and lunar climatic periodicities, which synchronise biological clocks. The question was how the animals can discriminate changing sunlight from moonlight intensities. For the first time, we could show a "moonlight window" in the larval ocelli of this midge, and propose a hypothesis on the underlying mechanisms. Driven by a lunar clock the image forming ocelli become transparent and convert during moonlit nights to a sensitive photometer, which can record the dynamics of environmental light. High resolution X-ray fluorescence (XRF) measurements of the distribution of trace minerals in single melanosomes combined with their fine structural details in various states of the lunar cycle provide a first insight into the enzymatic pathways for the generation of a dark melanin (like eumelanin) and a light coloured melanin (like phaeomelanin). Essential advantage of this approach is the spatial and temporal resolution of the metals associated with melanisation processes, which could never before be demonstrated in these details. The data may stimulate further research projects in biomedicine.

Parasitic-phase sea lampreys (Petromyzon marinus) are difficult to study in the wild. A series of laboratory studies (1984-1995) of single attacks on lake trout (Salvelinus namaycush), rainbow trout (Oncorhynchus mykiss), and burbot (Lota lota) examined host size selection; determined the effects of host size, host species, host strain, and temperature on host mortality; and estimated the weight of hosts killed per lamprey. Rainbow trout were more able and burbot less able to survive attacks than lake trout. Small sea lampreys actively selected the larger of two small hosts; larger sea lampreys attacked larger hosts in proportion to the hosts' body sizes, but actively avoided shorter hosts (a?? 600 mm) when larger were available. Host mortality was significantly less for larger (43-44%) than for smaller hosts (64%). However, the yearly loss of hosts per sea lamprey was less for small hosts (range, 6.8-14.2 kg per sea lamprey) than larger hosts (range, 11.4-19.3 kg per sea lamprey). Attacks at the lower of two temperature ranges (6.1-11.8A?C and 11.1-15.0A?C) did not significantly reduce the percentage of hosts killed (54% vs. 69%, p > 0.21), but longer attachment times at lower temperatures reduced the number of hosts attacked (33 vs. 45), and produced the lowest loss of hosts (6.6 kg per sea lamprey). Low temperature appeared to offset other factors that increase host mortality. Reanalysis of 789 attacks pooled from these studies, using forward stepwise logistic regression, also identified mean daily temperature as the dominant factor affecting host mortality. Observations in Lakes Superior, Huron, and Ontario support most laboratory results.

The dorsal lingual epithelium from the tongue of the toad Bufo marinus was mounted in an Ussing-type chamber, and the short-circuit current (I(sc)) was measured using a low-noise voltage clamp. With NaCl Ringer bathing the mucosal and serosal surfaces of the isolated tissue, an outwardly directed (mucosa-positive) I(sc) was measured that averaged -10.71+/-0.82 microA cm(-2) (mean +/- S.E.M., N=24) with a resistance of 615+/-152 Omega cm(2) (mean +/- S.E.M., N=10). Substitution of chloride with sulfate as the anion produced no significant change in I(sc). Fluctuation analysis with either NaCl or Na(2)SO(4) Ringer bathing both sides of the tissue revealed a spontaneous Lorentzian component, suggesting that the I(sc) was the result of K(+) secretion through spontaneously fluctuating channels in the apical membrane of the epithelium. This hypothesis was supported by the reversible inhibition of I(sc) by Ba(2+) added to the mucosal Ringer. Analysis of the kinetics of Ba(2+) inhibition of I(sc) indicates that there might be more than one type of K(+) channel carrying the I(sc). This hypothesis was supported by power spectra obtained with a serosa-to-mucosa K(+) gradient, which could be fitted to two Lorentzian components. At present, the K(+) secretory current cannot be localized to taste cells or other cells that might be associated with the secretion of saliva or mucus. Nonetheless, the resulting increase in [K(+)] in fluid bathing the mucosal surface of the tongue could presumably affect the sensitivity of the taste cells. These results contrast with those from the mammalian tongue, in which a mucosa-negative I(sc) results from amiloride-sensitive Na(+) transport.

Many of the far-reaching impacts of climate change on ecosystem function will be due to alterations in species interactions. However, our understanding of the effects of temperature on the dynamics of interactions between species is largely inadequate. Inducible defences persist in prey populations because defensive traits increase survival in the presence of predators but are costly when they are absent. Large-scale changes in the thermal climate are likely to alter the costs or benefits of these defences for ectotherms, whose physiological processes are driven by environmental temperature. A shift in costs of defensive traits would affect not only predator-prey interactions, but also the strength of selection for inducible defences in natural populations. We investigate the effect of temperature on the costs of behavioural defences in larvae of the marine toad, Rhinella marinus. Larvae were reared in the presence or absence of predator cues at both 25 and 30 °C. When exposed to predation cues, larvae reduced activity and spent less time feeding. Exposure to predation cues also reduced metabolic rate, presumably as a by-product of reducing activity levels. Larvae exposed to predation cues also grew more slowly, were smaller at metamorphosis and were poorer jumpers after metamorphosis--three traits associated with fitness in post-metamorphic anurans. We found that the costs of behavioural defences, in terms of larval growth, post-metamorphic size and jumping performance, were exacerbated at cooler temperatures. The thermal sensitivity of costs associated with defensive traits may explain geographic variation in plasticity of defensive traits in other species and suggests that changes in environmental temperature associated with climate change may affect predator-prey interactions in subtle ways not previously considered.

Living in the tidal zones of the sea requires synchronization with the dominant environmental influences of tidal, solar, and lunar periodicity. Endogenous clocks anticipate those geoclimatic changes and control the respective rhythms of vital functions. But the underlying mechanisms are only partly understood. While the circadian clocks in animals are investigated employing neurobiological, molecular, and genetic approaches, clocks with a lunar periodicity have been studied with reference to development and behavior only. Sites of their pacemakers, zeitgeber receptors, and coupled endocrine components are unknown. Here, a lunar-rhythmic change of shielding pigment transparency in the larval ocelli of the intertidal midge Clunio marinus is demonstrated for the first time as a possible access to the neurobiology of lunar timing mechanisms. We studied third instar larvae (Vigo strain) throughout the lunar cycle by light- and electron-microscopy as well as by x-ray fluorescence analysis for the identification of the pigment. Moonlight detection is a prerequisite for photic synchronization of the lunar clock. The larval ocelli of Clunio putatively may function as moonlight receptors and are also controlled by the circalunar clock itself, hence being primary candidates for tracing input and output pathways of the lunar pacemaker. Additionally, the demonstration of a reversible optical change of shielding pigment transparency in Clunio is a novel finding, not reported so far in any other animal species, and reveals a mechanism to enhance photosensitivity under the condition of very dim light. It represents a remarkable change of a sense organ from an imaging device to a radiometer. Its restriction to the developmental stage susceptible to lunar timing elucidates a unique sensory strategy evolved at the level of sensory input. It also raises basic questions about the biochemistry of optically active pigments, like melanin, and their intracellular control.

Background Many agricultural contaminants disrupt endocrine systems of wildlife. However, evidence of endocrine disruption in wild amphibians living in agricultural areas has been controversial. Typically, studies on the effects of pollutants on wildlife attempt to compare polluted with unpolluted sites. Objectives We took a novel approach to address this question by explicitly quantifying the relationship between gonadal abnormalities and habitats characterized by differing degrees of agricultural activity. Methods We quantified the occurrence of gonadal abnormalities and measures of gonadal function in at least 20 giant toads (Bufo marinus) from each of five sites that occur along a gradient of increasing agricultural land use from 0 to 97%. Results The number of abnormalities and frequency of intersex gonads increased with agriculture in a dose-dependent fashion. These gonadal abnormalities were associated with altered gonadal function. Testosterone, but not 17β-estradiol, concentrations were altered and secondary sexual traits were either feminized (increased skin mottling) or demasculinized (reduced forearm width and nuptial pad number) in intersex toads. Based on the end points we examined, female morphology and physiology did not differ across sites. However, males from agricultural areas had hormone concentrations and secondary sexual traits that were intermediate between intersex toads and non-agricultural male toads. Skin coloration at the most agricultural site was not sexually dimorphic; males had female coloration. Conclusions Steroid hormone concentrations and secondary sexual traits correlate with reproductive activity and success, so affected toads likely have reduced reproductive success. These reproductive abnormalities could certainly contribute to amphibian population declines occurring in areas exposed to agricultural contaminants. PMID:19057706

Ammocoetes of the anadromous sea lamprey Petromyzon marinus L. spend many years in freshwater before metamorphosing and migrating to sea. Metamorphosis involves the radical transformation from a substrate-dwelling, filter feeder into a free-swimming, parasitic feeder. In the present work we examined osmoregulatory differences between ammocoetes and transformers (metamorphic juveniles), and the effects of salinity acclimation. We measured the expression of key ion-transporting proteins [Na+/K+-ATPase, vacuolar (V)-type H+-ATPase and carbonic anhydrase (CA)] as well as a number of relevant blood parameters (hematocrit, [Na+] and [Cl -]). In addition, immunofluorescence microscopy was used to identify and characterize the distributions of Na+/K+-ATPase, V-type H+-ATPase and CA immunoreactive cells in the gill. Ammocoetes did not survive in the experiments with salinities greater than 10???, whereas survival in high salinity (???25-35???) increased with increased degree of metamorphosis in transformers. Plasma [Na+] and [Cl -] of ammocoetes in freshwater was lower than transformers and increased markedly at 10???. In transformers, plasma ions increased only at high salinity (>25???). Branchial Na+/K+-ATPase levels were ??? tenfold higher in transformers compared to ammocoetes and salinity did not affect expression in either group. However, branchial H +-ATPase expression showed a negative correlation with salinity in both groups. Na+/K+-ATPase immunoreactivity was strongest in transformers and associated with clusters of cells in the interlamellar spaces. H+-ATPase (B subunit) immunoreactivity was localized to epithelial cells not expressing high Na+/K+-ATPase immunoreactivity and having a similar tissue distribution as carbonic anhydrase. The results indicate that branchial Na+/K+-ATPase and salinity tolerance increase in metamorphosing lampreys, and that branchial H+-ATPase is downregulated by salinity.

The anaerobic cellulolytic rumen bacterium Eubacterium cellulosolvens produces a large array of cellulases and hemicellulases that are responsible for the hydrolysis of plant cell-wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises two tandemly repeated novel carbohydrate-binding modules (CBMs) and two catalytic domains belonging to glycoside hydrolase family 5 joined by flexible linker sequences. The novel CBM located at the N-terminus of the endoglucanase has been crystallized. The crystals belonged to the hexagonal space group P6(1)22 and contained a single molecule in the asymmetric unit. The structure of the L-selenomethionine derivative has been solved by a MAD experiment on crystals that diffracted to 1.75 Å resolution.

Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA. snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems. This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis. These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap. Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera. S. leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates. Thin-layer chromatography of S. leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E. coli immunoprecipitates. Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B. subtilis, as well. This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells. Images PMID:8458830

Arabinoxylan oligosaccharides (AXOS) are a promising class of prebiotics that have the potential to stimulate the growth of bifidobacteria and the production of butyrate in the human colon, known as the bifidogenic and butyrogenic effects, respectively. Although these dual effects of AXOS are considered beneficial for human health, their underlying mechanisms are still far from being understood. Therefore, this study investigated the metabolic interactions between Bifidobacterium longum subsp. longum NCC2705 (B. longum NCC2705), an acetate producer and arabinose substituent degrader of AXOS, and Eubacterium rectale ATCC 33656, an acetate-converting butyrate producer. Both strains belong to prevalent species of the human colon microbiota. The strains were grown on AXOS during mono- and coculture fermentations, and their growth, AXOS consumption, metabolite production, and expression of key genes were monitored. The results showed that the growth of both strains and gene expression in both strains were affected by cocultivation and that these effects could be linked to changes in carbohydrate consumption and concomitant metabolite production. The consumption of the arabinose substituents of AXOS by B. longum NCC2705 with the concomitant production of acetate allowed E. rectale ATCC 33656 to produce butyrate (by means of a butyryl coenzyme A [CoA]:acetate CoA-transferase), explaining the butyrogenic effect of AXOS. Eubacterium rectale ATCC 33656 released xylose from the AXOS substrate, which favored the B. longum NCC2705 production of acetate, explaining the bifidogenic effect of AXOS. Hence, those interactions represent mutual cross-feeding mechanisms that favor the coexistence of bifidobacterial strains and butyrate producers in the same ecological niche. In conclusion, this study provides new insights into the bifidogenic and butyrogenic effects of AXOS.

Arabinoxylan oligosaccharides (AXOS) are a promising class of prebiotics that have the potential to stimulate the growth of bifidobacteria and the production of butyrate in the human colon, known as the bifidogenic and butyrogenic effects, respectively. Although these dual effects of AXOS are considered beneficial for human health, their underlying mechanisms are still far from being understood. Therefore, this study investigated the metabolic interactions between Bifidobacterium longum subsp. longum NCC2705 (B. longum NCC2705), an acetate producer and arabinose substituent degrader of AXOS, and Eubacterium rectale ATCC 33656, an acetate-converting butyrate producer. Both strains belong to prevalent species of the human colon microbiota. The strains were grown on AXOS during mono- and coculture fermentations, and their growth, AXOS consumption, metabolite production, and expression of key genes were monitored. The results showed that the growth of both strains and gene expression in both strains were affected by cocultivation and that these effects could be linked to changes in carbohydrate consumption and concomitant metabolite production. The consumption of the arabinose substituents of AXOS by B. longum NCC2705 with the concomitant production of acetate allowed E. rectale ATCC 33656 to produce butyrate (by means of a butyryl coenzyme A [CoA]:acetate CoA-transferase), explaining the butyrogenic effect of AXOS. Eubacterium rectale ATCC 33656 released xylose from the AXOS substrate, which favored the B. longum NCC2705 production of acetate, explaining the bifidogenic effect of AXOS. Hence, those interactions represent mutual cross-feeding mechanisms that favor the coexistence of bifidobacterial strains and butyrate producers in the same ecological niche. In conclusion, this study provides new insights into the bifidogenic and butyrogenic effects of AXOS. PMID:26319874

Infection levels of eastern oysters by the unicellular pathogen Perkinsus marinus have been associated with anthropogenic influences in laboratory studies. However, these relationships have been difficult to investigate in the field because anthropogenic inputs are often associated with natural influences such as freshwater inflow, which can also affect infection levels. We addressed P. marinus-land use associations using field-collected data from Murrells Inlet, South Carolina, USA, a developed, coastal estuary with relatively minor freshwater inputs. Ten oysters from each of 30 reefs were sampled quarterly in each of 2 years. Distances to nearest urbanized land class and to nearest stormwater outfall were measured via both tidal creeks and an elaboration of Euclidean distance. As the forms of any associations between oyster infection and distance to urbanization were unknown a priori, we used data from the first and second years of the study as exploratory and confirmatory datasets, respectively. With one exception, quarterly land use associations identified using the exploratory dataset were not confirmed using the confirmatory dataset. The exception was an association between the prevalence of moderate to high infection levels in winter and decreasing distance to nearest urban land use. Given that the study design appeared adequate to detect effects inferred from the exploratory dataset, these results suggest that effects of land use gradients were largely insubstantial or were ephemeral with duration less than 3 months.

We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25. ppt) to 10 ??C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 ??C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7. ppt cultures acclimated to each temperature and then transferred to 3.5. ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30. days (3.5. ppt, 2 ??C: 0% viability), 60. days (3.5. ppt, 10 ??C: 0% viability) and 90. days (7. ppt, 2 ??C: 0.6 ?? 0.7%; 7. ppt, 10 ??C: 0.2 ?? 0.2%). ?? 2010 .

The sea lamprey, Petromyzon marinus, is an anadromous, semelparous species that is vulnerable to endangered in parts of its native range due in part to loss of spawning habitat because of man-made barriers. The ability of lampreys to return to the ocean or estuary and search out alternative spawning river systems would be limited by their osmoregulatory ability in seawater. A reduction in tolerance to salinity has been documented in migrants, although the underlying mechanisms have not been characterized. We examined the capacity for marine osmoregulation in upstream spawning migrants by characterizing the physiological effects of salinity challenge from a molecular perspective. Estuarine-captured migrants held in freshwater (FW) for ∼1 week (short-term acclimation) or 2 months (long-term acclimation) underwent an incremental salinity challenge until loss of equilibrium occurred and upper thresholds of 25 and 17.5, respectively, occurred. Regardless of salinity tolerance, all lamprey downregulated FW ion-uptake mechanisms [gill transcripts of Na+:Cl− cotransporter (NCC/slc12a3) and epithelial Na+ channel (ENaC/scnn1) and kidney Na+/K+-ATPase (NKA) protein and activity but not transcript]. At their respective salinity limits, lamprey displayed a clear osmoregulatory failure and were unable to regulate [Na+] and [Cl−] in plasma and intestinal fluid within physiological limits, becoming osmocompromised. A >90% drop in haematocrit indicated haemolysis, and higher plasma concentrations of the cytosolic enzymes alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase indicated damage to other tissues, including liver. However, >80% of short-term FW-acclimated fish were able to osmoregulate efficiently, with less haemolysis and tissue damage. This osmoregulatory ability was correlated with significant upregulation of the secretory form of Na+:K+:2Cl− cotransporter (NKCC1/slc12a2) transcript levels and the re-emergence of seawater

Experiments were performed to determine whether prostaglandins (PG) play a role in H+ and NH4+ excretion in the urinary bladder of Bufo marinus. Ten paired hemibladders from normal toads were mounted in chambers. One was control and the other hemibladder received PGE2 in the serosal medium (10(-5) M). H+ excretion was measured by change in pH in the mucosal fluid and reported in units of nmol (100 mg tissue)-1 (min)-1. NH4+ excretion was measured colorimetrically and reported in the same units. The control group H+ excretion was 8.4 +/- 1.67, while the experimental group was 16.3 +/- 2.64 (P less than 0.01). The NH4+ excretion in the experimental and control group was not significantly different. Bladders from toads in a 48-hr NH4+Cl acidosis (metabolic) did not demonstrate this response to PGE2 (P greater than 0.30). Toads were put in metabolic acidosis by gavaging with 10 ml of 120 mM NH4+Cl 3 x day for 2 days. In another experiment, we measured levels of PG in bladders from control (N) and animals placed in metabolic acidosis (MA). Bladders were removed from the respective toad, homogenized, extracted, and PG separated using high-pressure liquid chromatography and quantified against PG standards. The results are reported in ng (mg tissue)-1. PGE2 fraction in N was 1.09 +/- 0.14 and in MA was 3.21 +/- 0.63 (P less than 0.01). PGF1 alpha, F2 alpha and I2 were not significantly different in N and MA toads. Bladders were also removed from N and MA toads, and incubated in Ringer's solution containing (3H)arachidonic acid (0.2 microCi/ml) at 25 degrees C for 2 hr. Bladders were then extracted for PG and the extracts separated by thin layer chromatography. PG were identified using standards and autoradiography, scraped from plates, and counted in a scintillation detector. The results are reported in cpm/mg tissue x hr +/- SEM.

Dead lake trout (Salvelinus namaycush) killed by sea lamprey (Petromyzon marinus) were collected from the bottom of Lake Ontario using bottom trawls. The number of dead lake trout per hectare could be predicted from the number of type A-1 sea lamprey marks observed on live fish in September gillnet surveys (r2 = 0.60, P P > 0.05) from those of live fish with A-1 marks in 5 of 6 years where comparisons could be made. Compared with Lake Superior strain lake trout, Seneca Lake strain fish were only 0.41 times as likely to be attacked by sea lamprey and were less likely to die from an attack (both differences P < 0.05). Conservative estimates of the numbers of lake trout killed by sea lamprey in southern Lake Ontario from October to mid-November ranged from 17 000 in 1988 to 121 000 in 1984.

The role of the pineal complex in lamprey metamorphosis was investigated by examining the influence of pinealectomy and continuous light and darkness on the initiation of this event in anadromous sea lampreys, Petromyzon marinus L. Larval lampreys, which on the basis of a condition factor were considered likely to enter metamorphosis in July, were separated in May of 1979 and 1980 into the following groups: (1) intact controls, (2) sham-operated controls, (3) pinealectomized individuals, (4) those exposed to continuous light, and (5) those exposed to continuous light or dark. The importance of the pineal complex to metamorphosis was supported by morphological evidence that, in all presumably pinealectomized individuals that entered metamorphosis, the complex had apparently not been removed during the surgical procedure. The ways in which the pineal complex may be involved in lamprey metamorphosis are discussed.

In order to study the biological effects by bis(tributyltin) oxide (TBTO) exposure, chronic toxicity tests were conducted on the calanoid copepod Pseudodiaptomus marinus over two generations. The results indicated that nauplii were more sensitive than copepodites. F1 copepods were more vulnerable than F0 copepods and a drastic increase in mortality was observed as the TBTO concentration became higher. Exposure of copepods to 60 ng l -1 TBTO concentration reduced the fecundity and resulted in some females being infecund (in the F0 generation). The time to the first egg sac for females in the F1 generation exposed to 6 ng l -1 TBTO concentration was significantly reduced, and the fecundity of this generation was increased. The female-to-male ratio in the F1 generation exposed to 20 ng l -1 TBTO concentration was significantly reduced. These results show that the current ambient TBT concentration may influence populations of copepods in the coastal environment.

Polyphemusin is a broad-spectrum antimicrobial peptide isolated from hemocytes of the North American horseshoe crab Limulus polyphemus. To date the polyphemusin used for scientific analyses has been purified from the natural materials or obtained by chemical synthesis. We report here the recombinant expression in Escherichia coli, and subsequent purification, of a polyphemusin analogue (rLim1). To prevent toxicity of the antimicrobial peptide in the highly susceptible E. coli host, we used a carboxy-terminal fusion protein cloning strategy provided by a maltose-binding protein (MBP) gene fusion system (New England Biolabs). Antimicrobial activity of recombinant polyphemusin was similar to that seen with amidated native polyphemusin peptide. When rLim1 was tested for antibiotic activity against the apicomplexan protozoan oyster pathogen Perkinsus marinus, complete inhibition was observed at 12 micrograms/ml, and partial inhibition at 8 micrograms/ml.

The chemicals 3-trifluoromethyl-4-nitrophenol (TFM) or a combination of TFM and 2a??,5-dichloro-4a??-nitrosalicylanilide (Bayer 73) have been used to control the sea lamprey (Petromyzon marinus) in the Great Lakes for about 20 yr. These chemicals cause some mortalities of Oligochaeta and Hirudinea, immature forms of Ephemeroptera (Hexagenia sp.), and certain Trichoptera, Simuliidae, and Amphibia (Necturus sp.). The combination of TFM and Bayer 73 may affect some Pelecypoda and Gastropoda, but its overall effects on invertebrates are probably less than those of TFM alone. Granular Bayer 73 is likely to induce mortalities among oligochaetes, microcrustaceans, chironomids, and pelecypods. No evidence exists that the lampricides have caused the catastrophic decline or disappearance of any species. The overall impact of chemical control of sea lampreys on aquatic communities has been minor compared with the benefits derived.

The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (grdA) and the first 23 amino acids of the large subunit of protein PC of glycine reductase (grdC) belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklandii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.

Recent molecular analyses suggest that bacteria related to strains SS2/1 and SSC/2, previously reported to be distantly related to Anaerostipes caccae NCIMB 13811(T), represent one of the ten most abundant phylotypes detected in adult human faecal samples. These two strains were isolated as d-lactate-utilizing bacteria from faecal samples of a healthy individual. We show here that they share >99.9% similarity in 16S rRNA gene sequence with a new butyrate-producing isolate recovered from a colonic biopsy of a Crohn's disease patient, and also with the sequence reported recently for Eubacterium hadrum ATCC 29173(T). Biochemical profiling using API Rapid ID 32A and API ZYM test systems confirmed a close phenotypic similarity to E. hadrum ATCC 29173(T), but also indicated that the description of this species should be expanded to include the ability to produce butyrate from d-lactate and acetate. Phylogenetic analysis confirmed an affinity between E. hadrum and members of the genus Anaerostipes (92.3-94.2% sequence similarity) belonging to the family Lachnospiraceae (formerly Clostridium cluster XIVa). Based on phylogenetic, phenotypic and chemotaxonomic evidence it is proposed that E. hadrum be transferred to the genus Anaerostipes with the name Anaerostipes hadrus comb. nov. The type strain of A. hadrus comb. nov. is =ATCC 29173(T) (=DSM 3319(T) = VP 82-52(T)).

The mucus layer in the colon, acting as a barrier to prevent invasion of pathogens, is thinner and discontinuous in patients with ulcerative colitis (UC). A recent developed in vitro dynamic gut model, the M-SHIME, was used to compare long-term colonization of the mucin layer by the microbiota from six healthy volunteers (HV) and six UC patients and thus distinguish the mucin adhered from the luminal microbiota. Although under the same nutritional conditions, short-chain fatty acid production by the luminal communities from UC patients showed a tendency toward a lower butyrate production. A more in-depth community analysis of those microbial groups known to produce butyrate revealed that the diversity of the Clostridium coccoides/Eubacterium rectale and Clostridium leptum group, and counts of Faecalibacterium prausnitzii were lower in the luminal fractions of the UC samples. Counts of Roseburia spp. were lower in the mucosal fractions of the UC samples. qPCR analysis for butyryl-CoA:acetate CoA transferase, responsible for butyrate production, displayed a lower abundance in both the luminal and mucosal fractions of the UC samples. The M-SHIME model revealed depletion in butyrate producing microbial communities not restricted to the luminal but also in the mucosal samples from UC patients compared to HV.

The nucleotide sequence of the gene encoding the cellulose-binding protein B (CBPB) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3,429 nucleotides. The deduced amino acid sequence of CBPB contained one module highly similar to a catalytic module of glycosyl hydrolase family 9 (GHF9), one module partially similar to a family 3 carbohydrate-binding module (CBM3), two linkers, one module similar to a CBM of cellulose-binding protein A (CBPA) from E. cellulosolvens 5, and one module almost identical to a cell wall-binding module (CWBM) of CBPA. The module similar to GHF9 showed CMCase activity, and the modules similar to CBM3 and CBM of CBPA bound to cellulose. Moreover, the module highly similar to CWBM of CBPA bound to the cell walls prepared from E. cellulosolvens 5. The amino acid sequence of CBPB had a significant homology (64.15% sequence identity) with that of CBPA. These results suggest that cbpA and cbpB genes descended from the same ancestral cellulase gene.

As the Clostridium coccoides-Eubacterium rectale (Erec; clostridial phylogenetic cluster XIVa) group is one of the major groups of the human intestinal microbiota, DNA- and RNA-based population analysis techniques (denaturing gradient gel electrophoresis; DGGE) were developed and applied to assess the diversity and temporal stability (6 months-2 years) of this faecal clostridial microbiota in 12 healthy adults. The stability of the Erec group was compared with the stability of the predominant bacterial microbiota, which was also assessed with PCR-DGGE. In addition, the Erec group was quantified with a hybridization-based method. According to our results, the Erec group was diverse in each subject, but interindividual uniqueness was not as clear as that of the predominant bacteria. The Erec group was found to be temporally as stable as the predominant bacteria. Over 200 clones obtained from two samples proved the developed method to be specific. However, the amount of bacteria belonging to the Erec group was not related to the diversity of that same bacterial group. In conclusion, the newly developed DGGE method proved to be a valuable and specific tool for the direct assessment of the stability of the Erec group, demonstrating diversity in addition to short-term stability in most of the subjects studied.

2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) is the most abundant food-derived heterocyclic aromatic amine in well-cooked meats and may contribute to the recognized carcinogenicity of processed meats. In this study, a panel of human gut microbes was tested for their ability to convert PhIP to a conjugate PhIP-M1. Eubacterium hallii was newly identified to catalyse the conversion of PhIP to PhIP-M1 with high efficiency. The reaction was shown to involve the metabolism of glycerol to 3-hydroxypropionaldehyde as a key pathway. The proficiency of E. hallii in transforming PhIP in the presence of a complex intestinal microbiota was confirmed using batch fermentations inoculated with effluents from a continuous intestinal fermentation model mimicking human proximal and distal colon microbiota. In batch fermentations inoculated with proximal colon microbiota, PhIP-M1 transformation corresponded to an up to 300-fold increase of E. hallii. In contrast, PhIP transformation of distal colon microbiota was low but increased by 120-fold after supplementation with E. hallii. These findings indicate for the first time the relevance of the abundant commensal strict anaerobe E. hallii in the transformation of a dietary carcinogen that could contribute to its detoxification in the human colon.

We investigated the effect of CO(2) acidified sea water (S=35, 22 and 10(PSU)) on embryonic development of the intertidal amphipod Echinogammarus marinus (Leach). Low pH, but not low salinity (22(PSU)), resulted in a more protracted embryonic development in situ although the effect was only evident at low salinity. However reduced salinity, not pH, exerted a strong significant effect, on numbers and calcium content of hatchlings. Females exposed to low salinity (10(PSU)) did not carry eggs through to hatching. There was no significant difference in the number of viable hatchlings between females cultured in 22 and 35(PSU) but the exoskeleton of the juveniles at 22(PSU) contained significantly less calcium. Ocean acidification may affect aspects of E. marinus development but exposure to realistic low salinities appear, in the short term, to be more important in impacting development than exposure to CO(2) acidified sea water at levels predicted for 300 years time.

Recent genetic studies have shown that introgression rates among loci may greatly vary according to their location in the genome. In particular, several cases of mito-nuclear discordances have been reported for a wide range of organisms. In the present study, we examine the causes of discordance between mitochondrial (mtDNA) and nuclear DNA introgression detected in North American populations of the Great Black-backed Gull (Larus marinus), a Holarctic species, from the Nearctic North American Herring Gull (Larus smithsonianus). Our results show that extensive unidirectional mtDNA introgression from Larus smithsonianus into Larus marinus in North America cannot be explained by ancestral polymorphism but most likely results from ancient hybridization events occurring when Larus marinus invaded the North America. Conversely, our nuclear DNA results based on 12 microsatellites detected very little introgression from Larus smithsonianus into North American Larus marinus. We discuss these results in the framework of demographic and selective mechanisms that have been postulated to explain mito-nuclear discrepancies. We were unable to demonstrate selection as the main cause of mito-nuclear introgression discordance but cannot dismiss the possible role of selection in the observed pattern. Among demographic explanations, only drift in small populations and bias in mate choice in an invasive context may explain our results. As it is often difficult to demonstrate that selection may be the main factor driving the introgression of mitochondrial DNA in natural populations, we advocate that evaluating alternative demographic neutral hypotheses may help to indirectly support or reject hypotheses invoking selective processes.

Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe3O4) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe3O4) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus. Results show that MamC is located only in the magnetosome membrane of Mc. marinus. Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

Background The marine cyanobacterium Prochlorococcus is very abundant in warm, nutrient-poor oceanic areas. The upper mixed layer of oceans is populated by high light-adapted Prochlorococcus ecotypes, which despite their tiny genome (~1.7 Mb) seem to have developed efficient strategies to cope with stressful levels of photosynthetically active and ultraviolet (UV) radiation. At a molecular level, little is known yet about how such minimalist microorganisms manage to sustain high growth rates and avoid potentially detrimental, UV-induced mutations to their DNA. To address this question, we studied the cell cycle dynamics of P. marinus PCC9511 cells grown under high fluxes of visible light in the presence or absence of UV radiation. Near natural light-dark cycles of both light sources were obtained using a custom-designed illumination system (cyclostat). Expression patterns of key DNA synthesis and repair, cell division, and clock genes were analyzed in order to decipher molecular mechanisms of adaptation to UV radiation. Results The cell cycle of P. marinus PCC9511 was strongly synchronized by the day-night cycle. The most conspicuous response of cells to UV radiation was a delay in chromosome replication, with a peak of DNA synthesis shifted about 2 h into the dark period. This delay was seemingly linked to a strong downregulation of genes governing DNA replication (dnaA) and cell division (ftsZ, sepF), whereas most genes involved in DNA repair (such as recA, phrA, uvrA, ruvC, umuC) were already activated under high visible light and their expression levels were only slightly affected by additional UV exposure. Conclusions Prochlorococcus cells modified the timing of the S phase in response to UV exposure, therefore reducing the risk that mutations would occur during this particularly sensitive stage of the cell cycle. We identified several possible explanations for the observed timeshift. Among these, the sharp decrease in transcript levels of the dnaA gene

A Gram-negative bacterium, strain CZ-AM5(T), was isolated from an aerated biological filter in a marine recirculating aquaculture system in Tianjin, China. Its taxonomic position was investigated by using a polyphasic approach. Cells of strain CZ-AM5(T) were non-spore-forming rods, 0.5-0.8 µm wide and 1.2-2.0 µm long, and motile by means of one or two polar or lateral flagella. Strain CZ-AM5(T) was strictly aerobic, heterotrophic, oxidase-negative and catalase-positive. Growth occurred at 15-40 °C (optimum, 30-35 °C), at pH 6.5-10.5 (optimum, pH 7.0-7.5) and in the presence of 0-12.0 % (w/v) NaCl (optimum, 4.0 %). The predominant fatty acid was C18 : 1ω7c (80.3 %). Ubiquinone 10 (Q-10) was the sole respiratory quinone. The polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, an unknown aminolipid, an unknown phospholipid and three unknown lipids. The DNA G+C content was 60.4 mol%. Strain CZ-AM5(T) showed the highest 16S rRNA gene sequence similarity (96.5 %) to Phaeobacter caeruleus LMG 24369(T); it exhibited 16S rRNA gene sequence similarity of 95.0-96.5, 95.2-96.3, 96.2, 94.6-95.7 and 94.8-95.8 % to members of the genera Phaeobacter, Ruegeria, Citreimonas, Leisingera and Donghicola, respectively. However, phylogenetic trees based on 16S rRNA gene sequences showed that strain CZ-AM5(T) did not join any of the above genera, but formed a distinct lineage in the trees. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, strain CZ-AM5(T) is considered to represent a novel genus and species of the family Rhodobacteraceae, for which the name Cribrihabitans marinus gen. nov., sp. nov. is proposed. The type strain of Cribrihabitans marinus is CZ-AM5(T) ( = CGMCC 1.13219(T) = JCM 19401(T)).

We have discovered a new oligomeric protein component associated with the outer membrane of the ancestral eubacterium Thermotoga maritima. In electron micrographs, the protein, Omp alpha, appears as a rod-shaped spacer that spans the periplasm, connecting the outer membrane to the inner cell body. Purification, biochemical characterization and sequencing of Omp alpha suggest that it is a homodimer composed of two subunits of 380 amino acids with a calculated M(r) of 43,000 and a pI of 4.54. The sequence of the omp alpha gene indicates a tripartite organization of the protein with a globular NH2-terminal domain of 64 residues followed by a putative coiled-coil segment of 300 residues and a COOH-terminal, membrane-spanning segment. The predicted length of the coiled-coil segment (45 nm) correlates closely with the spacing between the inner and outer membranes. Despite sequence similarity to a large number of coiled-coil proteins and high scores in a coiled-coil prediction algorithm, the sequence of the central rod-shaped domain of Omp alpha does not have the typical 3.5 periodicity of coiled-coil proteins but rather has a periodicity of 3.58 residues. Such a periodicity was also found in the central domain of staphylococcal M protein and beta-giardin and might be indicative of a subclass of fibrous proteins with packing interactions that are distinct from the ones seen in other two-stranded coiled-coils. Images PMID:1330536

Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na(+) dependent. Consistent with the finding of a Na(+)-dependent Rnf complex is the presence of a conserved Na(+)-binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H2 + CO2. The energetic benefit may be one reason that butyrate is formed only from CO but not from H2 + CO2.

Recently, it was shown that the exposure to the potent hop phytoestrogen 8-prenylnaringenin (8-PN) depends on intestinal bacterial activation of isoxanthohumol (IX), but this occurs in only one-third of tested individuals. As the butyrate-producing Eubacterium limosum can produce 8-PN from IX, a probiotic strategy was applied to investigate whether 8-PN production could be increased in low 8-PN producers, thus balancing phytoestrogen exposure. Using fecal samples from high (Hop +) and low (Hop -) 8-PN-producing individuals, a Hop + and Hop - dynamic intestinal model was developed. In parallel, Hop + and Hop - human microbiota-associated rats were developed, germ-free (GF) rats acting as negative controls. IX and then IX + E. limosum were administered in the intestinal model and to the rats, and changes in 8-PN production and exposure were assessed. After dosing IX, 80% was converted into 8-PN in the Hop + model and highest 8-PN production, plasma concentrations, and urinary and fecal excretion occurred in the Hop + rats. Administration of the bacterium triggered 8-PN production in the GF rats and increased 8-PN production in the Hop - model and Hop - rats. 8-PN excretion was similar in the feces (294.1 +/- 132.2 nmol/d) and urine (8.5 +/- 1.1 nmol/d ) of all rats (n = 18). In addition, butyrate production increased in all rats. In conclusion, intestinal microbiota determined 8-PN production and exposure after IX intake. Moreover, E. limosum administration increased 8-PN production in low producers, resulting in similar 8-PN production in all rats.

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.

The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific "protein B"-complex of glycine reductase. The "protein B"-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)(-1) with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)(-1) in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep-tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.

Eubacterium pyruvativorans I-6(T) is a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source. This study investigated its metabolic properties and its likely ecological niche. Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate. No single amino acid supported growth, and peptides were required for growth on amino acids. Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation. Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate. l-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated. Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively. Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate. The metabolic strategy of E. pyruvativorans therefore resembles that of Clostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization. The fermentative niche of E. pyruvativorans appears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with C(2) units derived from other substrates to dispose of reducing equivalents.

Eubacterium limosum ZL-II was described to convert secoisolariciresinol (SECO) to its demethylating product 4,4'-dihydroxyenterodiol (DHEND) under anoxic conditions. However, the reaction cascade remains unclear. Here, the O-demethylase being responsible for the conversion was identified and characterized. Nine genes encoding two methyltransferase-Is (MT-I), two corrinoid proteins (CP), two methyltransferase-IIs (MT-II), and three activating enzymes (AE) were screened, cloned, and expressed in Escherichia coli. Four of the nine predicted enzymes, including ELI_2003 (MT-I), ELI_2004 (CP), ELI_2005 (MT-II), and ELI_0370 (AE), were confirmed to constitute the O-demethylase in E. limosum ZL-II. The complete O-demethylase (combining the four components) reaction system was reconstructed in vitro. As expected, the demethylating products 3-demethyl-SECO and DHEND were both produced. During the reaction process, ELI_2003 (MT-I) initially catalyzed the transfer of methyl group from SECO to the corrinoid of ELI_2004 ([Co(I)]-CP), yielding demethylating products and [CH3-Co(III)]-CP; then ELI_2005 (MT-II) mediated the transfer of methyl group from [CH3-Co(III)]-CP to tetrahydrofolate, forming methyltetrahydrofolate and [Co(I)]-CP. Due to the low redox potential of [Co(II)]/[Co(I)], [Co(I)]-CP was oxidized to [Co(II)]-CP immediately in vitro, and ELI_0370 (AE) was responsible for catalyzing the reduction of [Co(II)]-CP to its active form [Co(I)]-CP. The active-site residues in ELI_2003, ELI_2005, and ELI_0370 were subsequently determined using molecular modeling combined with site-directed mutagenesis. To our knowledge, this is the first study on the identification and characterization of a four-component O-demethylase from E. limosum ZL-II, which will facilitate the development of method to artificial synthesis of related bioactive chemicals.

Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na+ dependent. Consistent with the finding of a Na+-dependent Rnf complex is the presence of a conserved Na+-binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H2 + CO2. The energetic benefit may be one reason that butyrate is formed only from CO but not from H2 + CO2. PMID:25956767

Three strains of a Gram-positive, butyrate producing bacteria were isolated from the rumen content of grazing sheep and cow. The strains were anaerobic, Gram-positive cell wall, straight to slightly curved rod-shaped, non-spore-forming and single flagellate. C14:1, C14:0, C16:0 and C16:1 were the predominant fatty acids. The type of cell-wall peptidoglycan is A1γ. The DNA G+C content varied from 41.4 to 42.2 mol%. The 16S rRNA gene sequence similarities between the isolates and Eubacterium rectale, Roseburia hominis and Roseburia intestinalis were found to be 96, 95 and 95%, respectively. The phylogenetic tree showed that the strains constituted a different taxon, separate from other taxa with validly published names and forming a cluster with strains of Eubacterium rectale. On the basis of phenotypic, chemotaxonomic and phylogenetic results (16S RNA, DnaK, GroEL, atpA genes), isolates are considered to represent a novel species of a novel genus of the family Lachnospiraceae for which we propose the name Agathobacter ruminis gen. nov., sp. nov. with the type strain Agathobacter ruminis JK623T (=DSM 29029T =LMG 28559T). We also propose the transfer of Eubacterium rectale to the new genus Agathobacter gen. nov. This genus represents saccharoclastic chemoorganotrophic non-spore forming rods, with Gram-positive membrane, obligatory anaerobic. Main fermentation products on PYG medium were butyrate, acetate, hydrogen and lactate. Peptidoglycan in all species is of A1γ type. Type species is Agathobacter rectalis, gen. nov., comb nov. (Egghert 1935) with type strain ATCC 33656T (==KCTC 5835T). Two species of the new genus, Agathobacter rectalis and Agathobacter ruminis has been defined.

Sea lamprey (Petromyzon marinus) control in North America costs millions of dollars each year, and control measures are guided by assessment of lamprey-induced damage to fisheries. The favored prey of sea lamprey in freshwater ecosystems has been lake trout (Salvelinus namaycush). A key parameter in assessing sea lamprey damage, as well as managing lake trout fisheries, is the probability of an adult lake trout surviving a lamprey attack. The conventional value for this parameter has been 0.55, based on laboratory experiments. In contrast, based on catch curve analysis, mark-recapture techniques, and observed wounding rates, we estimated that adult lake trout in Lake Champlain have a 0.74 probability of surviving a lamprey attack. Although sea lamprey growth in Lake Champlain was lower than that observed in Lake Huron, application of an individual-based model to both lakes indicated that the probability of surviving an attack in Lake Champlain was only 1.1 times higher than that in Lake Huron. Thus, we estimated that lake trout survive a lamprey attack in Lake Huron with a probability of 0.66. Therefore, our results suggested that lethality of a sea lamprey attack on lake trout has been overestimated in previous model applications used in fisheries management. ?? 2008 NRC.

Recent studies proposed the use of chemosensory alarm cues to control the distribution of invasive sea lamprey Petromyzon marinus populations in the Laurentian Great Lakes and necessitate the evaluation of sea lamprey chemosensory alarm cues on valuable sympatric species such as white sucker. In two laboratory experiments, 10 replicate groups (10 animals each) of migratory white suckers were exposed to deionized water (control), conspecific whole-body extract, heterospecific whole-body extract (sea lamprey) and two potential predator cues (2-phenylethylamine HCl (PEA HCl) and human saliva) during the day, and exposed to the first four of the above cues at night. White suckers avoided the conspecific and the sea lamprey whole-body extract both during the day and at night to the same extent. Human saliva did not induce avoidance during the day. PEA HCl did not induce avoidance at a higher concentration during the day, or at night at the minimum concentration that was previously shown to induce maximum avoidance by sea lamprey under laboratory conditions. Our findings suggest that human saliva and PEA HCl may be potential species-specific predator cues for sea lamprey.

The light-driven inward chloride ion-pumping rhodopsin Nonlabens marinus rhodopsin-3 (NM-R3), from a marine flavobacterium, belongs to a phylogenetic lineage distinct from the halorhodopsins known as archaeal inward chloride ion-pumping rhodopsins. NM-R3 and halorhodopsin have distinct motif sequences that are important for chloride ion binding and transport. In this study, we present the crystal structure of a new type of light-driven chloride ion pump, NM-R3, at 1.58 Å resolution. The structure revealed the chloride ion translocation pathway and showed that a single chloride ion resides near the Schiff base. The overall structure, chloride ion-binding site, and translocation pathway of NM-R3 are different from those of halorhodopsin. Unexpectedly, this NM-R3 structure is similar to the crystal structure of the light-driven outward sodium ion pump, Krokinobacter eikastus rhodopsin 2. Structural and mutational analyses of NM-R3 revealed that most of the important amino acid residues for chloride ion pumping exist in the ion influx region, located on the extracellular side of NM-R3. In contrast, on the opposite side, the cytoplasmic regions of K. eikastus rhodopsin 2 were reportedly important for sodium ion pumping. These results provide new insight into ion selection mechanisms in ion pumping rhodopsins, in which the ion influx regions of both the inward and outward pumps are important for their ion selectivities.

Four behavioral experiments conducted in both the laboratory and the field provide evidence that adult sea lamprey (Petromyzon marinus) select spawning rivers based on the odor of larvae that they contain and that bile acids released by the larvae are part of this pheromonal odor. First, when tested in a recirculating maze, migratory adult lamprey spent more time in water scented with larvae. However, when fully mature, adults lost their responsiveness to larvae and preferred instead the odor of mature individuals. Second, when tested in a flowing stream, migratory adults swam upstream more actively when the water was scented with larvae. Third, when migratory adults were tested in a laboratory maze containing still water, they exhibited enhanced swimming activity in the presence of a 0.1 nM concentration of the two unique bile acids released by larvae and detected by adult lamprey. Fourth, when adults were exposed to this bile acid mixture within flowing waters, they actively swam into it. Taken together, these data suggest that adult lamprey use a bile acid based larval pheromone to help them locate spawning rivers and that responsiveness to this cue is influenced by current flow, maturity, and time of day. Although the precise identity and function of the larval pheromone remain to be fully elucidated, we believe that this cue will ultimately prove useful as an attractant in sea lamprey control.

Cloacal swabs and caecal contents sampled from 58 cane toads (Bufo marinus) in St George's parish, Grenada, during a 7-month period in 2011 were examined by an enrichment and selective culture method for presence of Salmonella spp. Twenty-four (41%) toads were positive for Salmonella spp. of which eight were Salmonella enterica serovar Javiana, and eight were S. enterica serovar Rubislaw. The other serovars were as follows: Montevideo, 6; Arechavaleta, 1; and serovar: IV:43:-:-, 1. The high frequency of isolation of serovar Javiana, an emerging human pathogen associated with several outbreaks in the recent years in the eastern United States, suggests a possible role for cane toads in transmission of this serovar. Although S. Rubislaw has been isolated from lizards, bats and cases of some human infections, there is no report of its carriage by cane toads, and in such high frequency. The rate of carriage of S. Montevideo, a cause for human foodborne outbreaks around the world was also over 10% in the 58 toads sampled in this study. The antimicrobial drug susceptibility tests against amoxicillin-clavulanic acid, ampicillin, cefotaxime, ceftazidime, ciprofloxacin, enrofloxacin, gentamicin, imipenem, nalidixic acid, streptomycin, tetracycline and trimethoprim-sulfamethoxazole showed that drug resistance is minimal and is of little concern. Antimicrobial resistance was limited to ampicillin and amoxicillin-clavulanic acid in one isolate of S. Javiana and one isolate of S. Rubislaw. This is the first report of isolation and antimicrobial susceptibilities of various Salmonella serovars not identified previously in cane toads in Grenada, West Indies.

Invasions by exotic organisms have had devastating affects on aquatic ecosystems, both ecologically and economically. One striking example of a successful invader that has dramatically affected fish community structure in freshwater lakes of North America is the sea lamprey (Petromyzon marinus). We used eight microsatellite loci and multiple analytical techniques to examine competing hypotheses concerning the origins and colonization history of sea lamprey (n = 741). Analyses were based on replicated invasive populations from Lakes Erie, Huron, Michigan, and Superior, populations of unknown origins from Lakes Ontario, Champlain, and Cayuga, and populations of anadromous putative progenitor populations in North America and Europe. Populations in recently colonized lakes were each established by few colonists through a series of genetic bottlenecks which resulted in lower allelic diversity in more recently established populations. The spatial genetic structure of invasive populations differed from that of native populations on the Atlantic coast, reflecting founder events and connectivity of invaded habitats. Anadromous populations were found to be panmictic (theta(P) = 0.002; 95% CI = -0.003-0.006; P > 0.05). In contrast, there was significant genetic differentiation between populations in the lower and upper Great Lakes (theta(P) = 0.007; P < 0.05; 95% CI = 0.003-0.009). Populations in Lakes Ontario, Champlain, and Cayuga are native. Alternative models that describe different routes and timing of colonization of freshwater habitats were examined using coalescent-based analyses, and demonstrated that populations likely originated from natural migrations via the St Lawrence River.

The complete nucleotide sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome has been determined. The lamprey genome is 16,201 bp in length and contains genes for 13 proteins, two rRNAs, 22 tRNAs and two major noncoding regions. The order and transcriptional polarities of protein-coding genes are basically identical to those of other chordate mtDNAs, demonstrating that the common mitochondrial gene organization of vertebrates was established at an early stage of vertebrate evolution. The two major noncoding regions are separated by two tRNA genes. The first region probably functions as the control region because it contains distinctive conserved sequence blocks (CSB-II and III) common to other vertebrate control regions. The central conserved domain observed in other vertebrate control regions is not found in the lamprey, suggesting that it is a recently evolved functional domain in vertebrates. Noncoding segments are not found in the expected position of the origin of replication for the second strand, suggesting either that one of the tRNA genes has a dual function or that the second noncoding region may function as the second-strand origin. The base composition at the wobble positions of fourfold degenerate codon families is highly biased toward thymine (32.7%). Values of GC-and AT-skew are typical of vertebrate mitochondrial genomes.genomes. PMID:7713438

The complete nucleotide sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome has been determined. The lamprey genome is 16,201 bp in length and contains genes for 13 proteins, two rRNAs, 22 tRNAs and two major noncoding regions. The order and transcriptional polarities of protein-coding genes are basically identical to those of other chordate mtDNAs, demonstrating that the common mitochondrial gene organization of vertebrates was established at early stage of vertebrate evolution. The two major noncoding regions are separated by two tRNA genes. The first region probably functions as the control region because it contains distinctive conserved sequence blocks (CSB-II and III) common to other vertebrate control regions. The central conserved domain observed in other vertebrate control regions is not found in the lamprey, suggesting that it is a recently evolved functional domain in vertebrates. Noncoding segments are not found in the expected position of the origin of replication for the second strand, suggesting either that one of the tRNA genes has a dual function or that the second noncoding region may function as the second-strand origin. The base composition at the wobble positions of fourfold degenerate codon families is highly biased toward thymine (32.7%). Values of GC- and AT-skew are typical of vertebrate mitochondrial genomes. 38 refs., 11 figs., 5 tabs.

A variety of unnatural bile acid derivatives (9a–9f) were synthesized and used to examine the specificity with which the sea lamprey (Petromyzon marinus) olfactory system detects these compounds. These compounds are analogs of petromyzonol sulfate (PS, 1), a component of the sea lamprey migratory pheromone. Both the stereochemical configuration at C5 (i.e., 5α vs. 5β) and the extent and sites of oxygenation (hydroxylation or ketonization) of the bile acid derived steroid skeleton were evaluated by screening the compounds for olfactory activity using electro-olfactogram recording. 5β-Petromyzonol sulfate (9a) elicited a considerable olfactory response at sub-nanomolar concentration. In addition, less oxygenated systems (i.e., 9b–9e) elicited olfactory responses, albeit with less potency. The sea lamprey sex pheromone mimic 9f (5β-3-ketopetromyzonol sulfate) was also examined and found to produce a much lower olfactory response. Mixture studies conducted with 9a and PS (1) suggest that stimulation is occurring via similar modes of activation, demonstrating a relative lack of specificity for recognition of the allo-configuration (i.e., 5α) in sea lamprey olfaction. This attribute could facilitate design of pheromone analogs to control this invasive species. PMID:21145335

The efficacy of 2’, 5-dichloro-4’-nitrosalicylanilide (niclosamide) at various concentrations and exposure times was tested against free-swimming larval sea lampreys (Petromyzon marinus) at 12°C and 17°C in Lake Huron water. Concentrations of niclosamide in test solutions ranged from 0.46 to 4.7 mg/L with pH 7.8 to 8.3, total alkalinity 78 to 88 mg/L as CaCO3, and total hardness 95 to 105 mg/L as CaCO3. In each test, six groups of larvae were exposed to a single concentration of niclosamide for times ranging from 30 s to 30 min. Exposure time was treated as the dose and, for each concentration tested, the exposure time necessary to kill 50 and 99.9% of larvae (ET50 and ET99.9) was determined. Linear regressions of the log10-transformed ET50 and ET99.9 on the log10-transformed niclosamide concentrations were significant at both temperatures with r2ranging from 0.94 to 0.98. The predicted ET50 ranged from 58 sec to 21.7 min and the ET99.9 ranged from 2.5 to 43.5 min across the concentrations and temperatures tested. Niclosamide required a significantly longer time to kill larvae at 12°C than at 17°C.

The sea lamprey Petromyzon marinus is an invasive pest in the Laurentian Great Lakes basin, threatening the persistence of important commercial and recreational fisheries. There is substantial interest in developing effective trapping practices via the application of behavior-modifying semiochemicals (odors). Here we report on the effectiveness of utilizing repellent and attractant odors in a push–pull configuration, commonly employed to tackle invertebrate pests, to improve trapping efficacy at permanent barriers to sea lamprey migration. When a half-stream channel was activated by a naturally derived repellent odor (a putative alarm cue), we found that sea lamprey located a trap entrance significantly faster than when no odor was present as a result of their redistribution within the stream. The presence of a partial sex pheromone, acting as an attractant within the trap, was not found to further decrease the time to when sea lamprey located a trap entrance relative to when the alarm cue alone was applied. Neither the application of alarm cue singly nor alarm cue and partial sex pheromone in combination was found to improve the numbers of sea lamprey captured in the trap versus when no odor was present — likely because nominal capture rate during control trials was unusually high during the study period. Behavioural guidance using these odors has the potential to both improve control of invasive non-native sea lamprey in the Great Lakes as well as improving the efficiency of fish passage devices used in the restoration of threatened lamprey species elsewhere.

Sea lampreys Petromyzon marinus, an invasive pest in the Upper Great Lakes, avoid odours that represent danger in their habitat. These odours include conspecific alarm cues and predator cues, like 2-phenylethylamine hydrochloride (PEA HCl), which is found in the urine of mammalian predators. Whether conspecific alarm cues and predator cues function additively or synergistically when mixed together is unknown. The objectives of this experimental study were to determine if the avoidance response of sea lamprey to PEA HCl is proportional to the concentration delivered, and if the avoidance response to the combination of a predator cue (PEA HCl) and sea lamprey alarm cue is additive. To accomplish the first objective, groups of ten sea lampreys were placed in an artificial stream channel and presented with stepwise concentrations of PEA HCl ranging from 5 × 10−8 to 5 × 10−10 M and a deionized water control. Sea lampreys exhibited an increase in their avoidance behaviour in response to increasing concentrations of PEA HCl. To accomplish the second objective, sea lampreys were exposed to PEA HCl, conspecific alarm cue and a combination of the two. Sea lampreys responded to the combination of predator cue and conspecific alarm cue in an additive manner.

A Gram-negative, proteorhodopsin-containing, orange pigmented, rod-shaped and strictly aerobic bacterium, designated strain AKS622(T), was isolated from a glacier core collected from the coast of King George Island, Antarctica. 16S rRNA gene sequence analysis revealed that strain AKS622(T) was affiliated to the genus Nonlabens of the family Flavobacteriaceae and showed highest similarity to Nonlabens marinus S1-08(T) (97.9%). The level of DNA-DNA relatedness between strain AKS622(T) and N. marinus S1-08(T) was 46%. Optimal growth of strain AKS622(T) was observed at pH 7.0, at 15 °C and with 2.0% NaCl. The predominant cellular fatty acids were anteiso-C(15 : 0), iso-C(16 : 0), iso-C(16 : 0) 3-OH, C17:0 2-OH and summed feature 3 (comprising C(16 : 1)ω7c and/or C(16 : 1)ω6c). The DNA G+C content was 37.9 mol%. The major respiratory quinone was MK-6. Phosphatidylethanolamine, four unidentified glycolipids, three unidentified aminolipids and one unidentified lipid were detected as major polar lipids. On the basis of the data from this polyphasic taxonomic study, it was concluded that strain AKS622(T) represents a novel species within the genus Nonlabens, for which the name Nonlabens antarcticus sp. nov. is proposed. The type strain is AKS622(T) ( = KCCM 43019(T) = JCM 14068(T)). Emended descriptions of N. marinus Park et al. 2012 and Nonlabens agnitus Yi and Chun 2012 are given.

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine

Coenzyme B(12)-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacterium barkeri catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins with mutations in the highly conserved coenzyme binding-motif DXH(X)(2)G(X)(41)GG (D483N and H485Q) exhibited decreased substrate turnover by 2000-fold and >4000-fold, respectively. These findings are consistent with the notion of H485 hydrogen-bonded to D483 being the lower axial ligand of adenosylcobalamin in 2-methyleneglutarate mutase. (E)- and (Z)-2-methylpent-2-enedioate and all four stereoisomers of 1-methylcyclopropane-1,2-dicarboxylate were synthesized and tested, along with acrylate, with respect to their inhibitory potential. Acrylate and the 2-methylpent-2-enedioates were noninhibitory. Among the 1-methylcyclopropane-1,2-dicarboxylates only the (1R,2R)-isomer displayed weak inhibition (noncompetitive, K(i) = 13 mM). Short incubation (5 min) of 2-methyleneglutarate mutase with 2-methyleneglutarate under anaerobic conditions generated an electron paramagnetic resonance (EPR) signal (g(xy) approximately 2.1; g(z) approximately 2.0), which by analogy with the findings on glutamate mutase from Clostridium cochlearium [Biochemistry, 1998, 37, 4105-4113] was assigned to cob(II)alamin coupled to a carbon-centered radical. At longer incubation times (>1 h), inactivation of the mutase occurred concomitant with the formation of oxygen-insensitive cob(II)alamin (g(xy) approximately 2.25; g(z) approximately 2.0). In order to identify the carbon-centered radical, various (13)C- and one (2)H-labeled substrate/product molecules were synthesized. Broadening (0.5 mT) of the EPR signal around g = 2.1 was observed only when C2 and/or C4 of 2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5'-(13)C]adenosylcobalamin was used as coenzyme. The inhibition and EPR data are discussed in the context of the addition-elimination and fragmentation-recombination mechanisms

Spermiating male sea lamprey (Petromyzon marinus) release a sex pheromone, of which a component, 7α, 12α, 24-trihydoxy-3-one-5α-cholan-24-sulfate (3kPZS), has been identified and shown to induce long distance preference responses in ovulated females. However, other pheromone components exist, and when 3kPZS alone was used to control invasive sea lamprey populations in the Laurentian Great Lakes, trap catch increase was significant, but gains were generally marginal. We hypothesized that free-ranging sea lamprey populations discriminate between a partial and complete pheromone while migrating to spawning grounds and searching for mates at spawning grounds. As a means to test our hypothesis, and to test two possible uses of sex pheromones for sea lamprey control, we asked whether the full sex pheromone mixture released by males (spermiating male washings; SMW) is more effective than 3kPZS in capturing animals in traditional traps (1) en route to spawning grounds and (2) at spawning grounds. At locations where traps target sea lampreys en route to spawning grounds, SMW-baited traps captured significantly more sea lampreys than paired 3kPZS-baited traps (~10 % increase). At spawning grounds, no difference in trap catch was observed between 3kPZS and SMW-baited traps. The lack of an observed difference at spawning grounds may be attributed to increased pheromone competition and possible involvement of other sensory modalities to locate mates. Because fishes often rely on multiple and sometimes redundant sensory modalities for critical life history events, the addition of sex pheromones to traditionally used traps is not likely to work in all circumstances. In the case of the sea lamprey, sex pheromone application may increase catch when applied to specifically designed traps deployed in streams with low adult density and limited spawning habitat.

Digestion affects acid-base status, because the net transfer of HCl from the blood to the stomach lumen leads to an increase in HCO3(-) levels in both extra- and intracellular compartments. The increase in plasma [HCO3(-)], the alkaline tide, is particularly pronounced in amphibians and reptiles, but is not associated with an increased arterial pH, because of a concomitant rise in arterial PCO2 caused by a relative hypoventilation. In this study, we investigate whether the postprandial increase in PaCO2 of the toad Bufo marinus represents a compensatory response to the increased plasma [HCO3(-)] or a state-dependent change in the control of pulmonary ventilation. To this end, we successfully prevented the alkaline tide, by inhibiting gastric acid secretion with omeprazole, and compared the response to that of untreated toads determined in our laboratory during the same period. In addition, we used vascular infusions of bicarbonate to mimic the alkaline tide in fasting animals. Omeprazole did not affect blood gases, acid-base and haematological parameters in fasting toads, but abolished the postprandial increase in plasma [HCO3(-)] and the rise in arterial PCO2 that normally peaks 48 h into the digestive period. Vascular infusion of HCO3(-), that mimicked the postprandial rise in plasma [HCO3(-)], led to a progressive respiratory compensation of arterial pH through increased arterial PCO2. Thus, irrespective of whether the metabolic alkalosis is caused by gastric acid secretion in response to a meal or experimental infusion of bicarbonate, arterial pH is being maintained by an increased arterial PCO2. It seems, therefore, that the elevated PCO2, occuring during the postprandial period, constitutes of a regulated response to maintain pH rather than a state-dependent change in ventilatory control.

Circadian rhythms pre-adapt the physiology of most organisms to predictable daily changes in the environment. Some marine organisms also show endogenous circalunar rhythms. The genetic basis of the circalunar clock and its interaction with the circadian clock is unknown. Both clocks can be studied in the marine midge Clunio marinus (Chironomidae, Diptera), as different populations have different local adaptations in their lunar and diurnal rhythms of adult emergence, which can be analyzed by crossing experiments. We investigated the genetic basis of population variation in clock properties by constructing the first genetic linkage map for this species, and performing quantitative trait locus (QTL) analysis on variation in both lunar and diurnal timing. The genome has a genetic length of 167-193 centimorgans based on a linkage map using 344 markers, and a physical size of 95-140 megabases estimated by flow cytometry. Mapping the sex determining locus shows that females are the heterogametic sex, unlike most other Chironomidae. We identified two QTL each for lunar emergence time and diurnal emergence time. The distribution of QTL confirms a previously hypothesized genetic basis to a correlation of lunar and diurnal emergence times in natural populations. Mapping of clock genes and light receptors identified ciliary opsin 2 (cOps2) as a candidate to be involved in both lunar and diurnal timing; cryptochrome 1 (cry1) as a candidate gene for lunar timing; and two timeless (tim2, tim3) genes as candidate genes for diurnal timing. This QTL analysis of lunar rhythmicity, the first in any species, provides a unique entree into the molecular analysis of the lunar clock.

Sea lamprey Petromyzon marinus control in the Great Lakes primarily involves application of lampricides to streams where larval production occurs to kill larvae prior to their metamorphosing and entering the lakes as parasites (juveniles). Because lampricides are not 100% effective, larvae that survive treatment maymetamorphose before streams are again treated. Larvae that survive treatment have not beenwidely studied, so their dynamics are notwell understood.Wetagged and released larvae in six Great Lake tributaries following lampricide treatment and estimated vital demographic rates using multistate tag-recovery models. Model-averaged larval survivals ranged from 56.8 to 57.6%. Model-averaged adult recovery rates, which were the product of juvenile survivals and adult capture probabilities, ranged from 6.8 to 9.3%. Using stochastic simulations, we estimated production of juvenile sea lampreys from a hypothetical population of treatment survivors under different growth conditions based on parameter estimates from this research. For fast-growing populations, juvenile production peaked 2 years after treatment. For slow-growing populations, juvenile production was approximately one-third that of fast-growing populations,with production not peaking until 4 years after treatment. Our results suggest that dynamics (i.e., survival, metamorphosis) of residual larval populations are very similar to those of untreated larval populations. Consequently, residual populations do not necessarily warrant special consideration for the purpose of sea lamprey control and can be ranked for treatment along with other populations. Consecutive lampricide treatments, which are under evaluation by the sea lamprey control program, would bemost effective for reducing juvenile production in large, fast-growing populations.

Circadian rhythms pre-adapt the physiology of most organisms to predictable daily changes in the environment. Some marine organisms also show endogenous circalunar rhythms. The genetic basis of the circalunar clock and its interaction with the circadian clock is unknown. Both clocks can be studied in the marine midge Clunio marinus (Chironomidae, Diptera), as different populations have different local adaptations in their lunar and diurnal rhythms of adult emergence, which can be analyzed by crossing experiments. We investigated the genetic basis of population variation in clock properties by constructing the first genetic linkage map for this species, and performing quantitative trait locus (QTL) analysis on variation in both lunar and diurnal timing. The genome has a genetic length of 167–193 centimorgans based on a linkage map using 344 markers, and a physical size of 95–140 megabases estimated by flow cytometry. Mapping the sex determining locus shows that females are the heterogametic sex, unlike most other Chironomidae. We identified two QTL each for lunar emergence time and diurnal emergence time. The distribution of QTL confirms a previously hypothesized genetic basis to a correlation of lunar and diurnal emergence times in natural populations. Mapping of clock genes and light receptors identified ciliary opsin 2 (cOps2) as a candidate to be involved in both lunar and diurnal timing; cryptochrome 1 (cry1) as a candidate gene for lunar timing; and two timeless (tim2, tim3) genes as candidate genes for diurnal timing. This QTL analysis of lunar rhythmicity, the first in any species, provides a unique entree into the molecular analysis of the lunar clock. PMID:22384150

The rumen anaerobic cellulolytic bacterium Eubacterium cellulosolvens produces a large range of cellulases and hemicellulases responsible for the efficient hydrolysis of plant cell wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises a tandemly repeated carbohydrate-binding module (CBM65) fused to a glycoside hydrolase family 5 (Cel5A) catalytic domain, joined by flexible linker sequences. The second carbohydrate-binding module located at the C-terminus side of the endoglucanase (CBM65B) has been co-crystallized with either cellohexaose or xyloglucan heptasaccharide. The crystals belong to the hexagonal space group P6(5) and tetragonal space group P4(3)2(1)2, containing a single molecule in the asymmetric unit. The structures of CBM65B have been solved by molecular replacement.

In coastal Louisiana, the development of large-scale freshwater diversion projects has led to controversy over their effects on oyster resources. Using controlled laboratory experiments in combination with a field study, we examined the effects of pulsed freshwater events (freshet) of different magnitude, duration, and rate of change on oyster resources. Laboratory and field evidence indicate that low salinity events (<5 psu) decreased Perkinsus marinus infection intensities. Furthermore, when salinity was low (<5 psu), parasite infection intensities continued to decrease even as temperatures exceeded 20°C. At the same time, oyster growth was positively correlated with salinity. To maximize oyster production, data indicate that both low and high salinity events will be necessary.

Specific dynamic action (SDA), the accumulated energy expended on all physiological processes associated with meal digestion, is strongly influenced by features of both the meal and the organism. We assessed the effects of meal size, meal type, body temperature, and body size on the postprandial metabolic response and calculated SDA of the marine toad, Bufo marinus. Peak postprandial rates of O(2) consumption (.V(O2)) and CO(2) production (.V(CO2)) and SDA increased with meal size (5%-20% of body mass). Postprandial metabolism was impacted by meal type; the digestion of hard-bodied superworms (Zophobas larva) and crickets was more costly than the digestion of soft-bodied earthworms and juvenile rats. An increase in body temperature (from 20 degrees to 35 degrees C) altered the postprandial metabolic profile, decreasing its duration and increasing its magnitude, but did not effect SDA, with the cost of meal digestion remaining constant across body temperatures. Allometric mass exponents were 0.69 for standard metabolic rate, 0.85 for peak postprandial .V(O2), and 1.02 for SDA; therefore, the factorial scope of peak postprandial .V(O2) increased with body mass. The mass of nutritive organs (stomach, liver, intestines, and kidneys) accounted for 38% and 20% of the variation in peak postprandial .V(O2) and SDA, respectively. Toads forced to exercise experienced 25-fold increases in .V(O2) much greater than the 5.5-fold increase experience during digestion. Controlling for meal size, meal type, and body temperature, the specific dynamic responses of B. marinus are similar to those of the congeneric Bufo alvarius, Bufo boreas, Bufo terrestris, and Bufo woodhouseii.

Lake-wide abundance of spawning-phase sea lampreys (Petromyzon marinus) can be used as one means to evaluate sea lamprey control efforts in the Great Lakes. Lake-wide abundance in each Great Lake was the sum of estimates for all streams thought to contribute substantial numbers of sea lampreys. A subset of these streams was sampled with traps and mark-recapture studies were conducted. When sea lampreys were captured in traps, but no mark-recapture study was conducted, abundance was estimated from a relation between trap catch and mark-recapture estimates observed in other years. In non-sampled streams, a regression model that used stream drainage area, geographic region, larval sea lamprey, production potential, the number of years since the last lampricide treatment, and spawning year was used to predict abundance of spawning-phase sea lampreys. The combination of estimates from sampled and non-sampled streams provided a 20-year time series of spawning-phase sea lamprey abundance estimates in the Great Lakes.

This study examined olfactory sensory neuron morphology and physiological responsiveness in newly hatched sea lamprey, Petromyzon marinus L. These prolarvae hatch shortly after neural tube formation, and stay within nests for approximately 18 days, before moving downstream to silty areas where they burrow, feed and pass to the larval stage. To explore the possibility that the olfactory system is functioning during this prolarval stage, morphological and physiological development of olfactory sensory neurons was examined. The nasal cavity contained an olfactory epithelium with ciliated olfactory sensory neurons. Axons formed aggregates in the basal portion of the olfactory epithelium and spanned the narrow distance between the olfactory epithelium and the brain. The presence of asymmetric synapses with agranular vesicles within fibers in the brain, adjacent to the olfactory epithelium suggests that there was synaptic connectivity between olfactory sensory axons and the brain. Neural recordings from the surface of the olfactory epithelium showed responses following the application of L-arginine, taurocholic acid, petromyzonol sulfate (a lamprey migratory pheromone), and water conditioned by conspecifics. These results suggest that lampreys may respond to olfactory sensory input during the prolarval stage. ?? 2006 Springer Science + Business Media, LLC.

Lamprey is one of only two living jawless vertebrates, a group that includes the first vertebrates. Comparisons between lamprey and jawed vertebrates have yielded important insights into the origin and evolution of vertebrate physiology, morphology and development. Despite its key phylogenetic position, studies of lamprey have been limited by their complex life history, which makes traditional genetic approaches impossible. The CRISPR/Cas9 system is a bacterial defense mechanism that was recently adapted to achieve high-efficiency targeted mutagenesis in eukaryotes. Here we report CRISPR/Cas9-mediated disruption of the genes Tyrosinase and FGF8/17/18 in the sea lamprey Petromyzon marinus, and detail optimized parameters for producing mutant F0 embryos. Using phenotype and genotype analyses, we show that CRISPR/Cas9 is highly effective in the sea lamprey, with a majority of injected embryos developing into complete or partial mutants. The ability to create large numbers of mutant embryos without inbred lines opens exciting new possibilities for studying development in lamprey and other non-traditional model organisms with life histories that prohibit the generation of mutant lines. PMID:26511928

Successful implementation of a sea lamprey (Petromyzon marinus) control technique that uses sterilized males to reduce reproduction presently depends on the importation of large numbers of males outside of the target population. Strategies were examined for releasing male sea lampreys from Lakes Michigan and Huron into the Lake Superior spawning population and the ability of these introduced males to compete with resident males and spawn with resident females. During 1987, 553 (9%) of 6,324 imported fertile males released at 12 shoreline and one offshore site in Lake Superior were recaptured. Most remained within 20 km of the release site and entered the first stream encountered. During 1988, 393 (18%) of 2,208 imported fertile males released directly into three spawning rivers were recaptured. In both cases, animals released early during the spawning run were more likely to be recaptured than those released later. Introduced males successfully competed with resident males and spawned with resident females. Demonstrating that male sea lampreys could reproduce successfully when relocated supported subsequent large-scale field trials of the sterile-male-release technique.

Thyroid hormones (THs) are crucial for normal vertebrate development and are the one obligate morphogen that drives amphibian metamorphosis. However, contrary to other metamorphosing vertebrates, lampreys exhibit a sharp drop in serum TH early in metamorphosis, and anti-thyroid agents such as potassium perchlorate (KClO(4)) induce metamorphosis. The type 2 deiodinase (D2) enzyme is a key regulator of TH availability during amphibian metamorphosis. We set out to determine how D2 may be involved in the regulation of lamprey metamorphosis and thyroid homeostasis. We cloned a 1.8Kb Petromyzon marinus D2 cDNA that includes the entire protein coding region and a selenocysteine (Sec) codon. Northern blotting indicated that the lamprey D2 mRNA is the longest reported to date (>9Kb). Using real-time PCR, we showed that intestinal and hepatic D2 mRNA levels were elevated prior to and during the early stages of metamorphosis and then declined dramatically to low levels that were sustained for the remainder of metamorphosis. These data are consistent with previously reported changes in serum TH levels and deiodinase activity. Treatment of larvae with either TH or KClO(4) significantly affected D2 mRNA levels in the intestine and liver. These D2 mRNA levels during metamorphosis and in response to thyroid challenges suggest that D2 may function in the regulation of TH levels during lamprey metamorphosis and the maintenance of TH homeostasis.

Lamprey is one of only two living jawless vertebrates, a group that includes the first vertebrates. Comparisons between lamprey and jawed vertebrates have yielded important insights into the origin and evolution of vertebrate physiology, morphology and development. Despite its key phylogenetic position, studies of lamprey have been limited by their complex life history, which makes traditional genetic approaches impossible. The CRISPR/Cas9 system is a bacterial defense mechanism that was recently adapted to achieve high-efficiency targeted mutagenesis in eukaryotes. Here we report CRISPR/Cas9-mediated disruption of the genes Tyrosinase and FGF8/17/18 in the sea lamprey Petromyzon marinus, and detail optimized parameters for producing mutant F0 embryos. Using phenotype and genotype analyses, we show that CRISPR/Cas9 is highly effective in the sea lamprey, with a majority of injected embryos developing into complete or partial mutants. The ability to create large numbers of mutant embryos without inbred lines opens exciting new possibilities for studying development in lamprey and other non-traditional model organisms with life histories that prohibit the generation of mutant lines.

The sea lamprey Petromyzon marinus (Linnaeus) is both an invasive non-native species in the Laurentian Great Lakes of North America and an imperiled species in much of its native range in North America and Europe. To compare and contrast how understanding of population ecology is useful for control programs in the Great Lakes and restoration programs in Europe, we review current understanding of the population ecology of the sea lamprey in its native and introduced range. Some attributes of sea lamprey population ecology are particularly useful for both control programs in the Great Lakes and restoration programs in the native range. First, traps within fish ladders are beneficial for removing sea lampreys in Great Lakes streams and passing sea lampreys in the native range. Second, attractants and repellants are suitable for luring sea lampreys into traps for control in the Great Lakes and guiding sea lamprey passage for conservation in the native range. Third, assessment methods used for targeting sea lamprey control in the Great Lakes are useful for targeting habitat protection in the native range. Last, assessment methods used to quantify numbers of all life stages of sea lampreys would be appropriate for measuring success of control in the Great Lakes and success of conservation in the native range.

The sea lamprey (Petromyzon marinus) invaded the upper Laurentian Great Lakes and feeds on valued fish. The Cheboygan River, Michigan, USA, is a large sea lamprey producing tributary to Lake Huron and despite having a renovated dam 2 km from the river mouth that presumably blocks sea lamprey spawning migrations, the watershed upstream of the dam remains infested with larval sea lamprey. A navigational lock near the dam has been hypothesized as the means of escapement of adult sea lampreys from Lake Huron and source of the upper river population (H1). However, an alternative hypothesis (H2) is that some sea lampreys complete their life cycle upstream of the dam, without entering Lake Huron. To evaluate the alternative hypothesis, we gathered angler reports of lamprey wounds on game fishes upstream of the dam, and captured adult sea lampreys downstream and upstream of the dam to contrast abundance, run timing, size, and statolith microchemistry. Results indicate that a small population of adult sea lampreys (n < 200) completed their life cycle upstream of the dam during 2013 and 2014. This is the most comprehensive evidence that sea lampreys complete their life history within a tributary of the upper Great Lakes, and indicates that similar landlocked populations could occur in other watersheds. Because the adult sea lamprey population upstream of the dam is small, complete elimination of the already low adult escapement from Lake Huron might allow multiple control tactics such as lampricides, trapping, and sterile male release to eradicate the population.

The ability to predict when sea lampreys (Petromyzon marinus) will metamorphose from the larval phase to the parasitic phase is essential to the operation of the sea lamprey control program. During the spring of 1994, two populations of sea lamprey larvae from two rivers were captured, measured, weighed, implanted with coded wire tags, and returned to the same sites in the streams from which they were taken. Sea lampreys were recovered in the fall, after metamorphosis would have occurred, and checked for the presence of a tag. When the spring data were compared to the fall data it was found that the minimum requirements (length ≥ 120 mm, weight ≥ 3 g, and condition factor ≥ 1.50) suggested for metamorphosis did define a pool of larvae capable of metamorphosing. However, logistic regressions that relate the probability of metamorphosis to size are necessary to predict metamorphosis in a population. The data indicated, based on cross-validation, that weight measurements alone predicted metamorphosis with greater precision than length or condition factor in both the Marengo and Amnicon rivers. Based on the Akaike Information Criterion, weight alone was a better predictor in the Amnicon River, but length and condition factor combined predicted metamorphosis better in the Marengo River. There would be no additional cost if weight alone were used instead of length. However, if length and weight were measured the gain in predictive power would not be enough to justify the additional cost.

The primary method of sea lamprey (Petromyzon marinus) control in the Great Lakes is the treatment of streams and rivers with the pesticide 3-trifluoromethyl-4-nitrophenol (TFM), which targets larval sea lamprey. However, less is known about the effects of TFM on other stages of the sea lamprey's complex life cycle. The goal of this study was to determine how TFM affected internal energy stores, metabolites, and ion balance in larval, juvenile (parasitic) and adult sea lamprey. The larvae were more tolerant to TFM than the adults, with a 2-fold higher 12h TFM LC50 and a 1.5-fold higher LC99.9. Acute (3h) exposure of the larvae, parasites and adults to their respective 12h TFM LC99.9 led to marked reductions in glycogen and phosphocreatine in the adult brain, with lesser or no effect in the larvae and parasites. Increased lactate in the brain, at less than the expected stoichiometry, suggested that it was exported to the blood. Kidney glycogen declined after TFM exposure, suggesting that this organ plays an important role in glucose homeostasis. TFM-induced disturbances to ion balance were minimal. In conclusion, TFM perturbs energy metabolism in all major stages of the sea lamprey life cycle in a similar fashion, but the adults appear to be the most sensitive. Thus, the adult stage could be a viable and effective target for TFM treatment, particularly when used in combination with other existing and emerging strategies of sea lamprey control.

A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2 Images PMID:6758698

Two novel obligately anaerobic, Gram-stain-positive, saccharolytic and non-proteolytic spore-forming bacilli (strains CD3:22(T) and N1(T)) are described. Strain CD3:22(T) was isolated from a biopsy of the small intestine of a child with coeliac disease, and strain N1(T) from the saliva of a healthy young man. The cells of both strains were observed to be filamentous, approximately 5 to >20 µm long, some of them curving and with swellings. The novel organisms produced H(2)S, NH(3), butyric acid and acetic acid as major metabolic end products. Phylogenetic analyses, based on comparative 16S rRNA gene sequencing, revealed close relationships (98% sequence similarity) between the two isolates, as well as the type strain of Eubacterium saburreum and four other Lachnospiraceae bacterium-/E. saburreum-like organisms. This group of bacteria were clearly different from any of the 19 known genera in the family Lachnospiraceae. While Eubacterium species are reported to be non-spore-forming, reanalysis of E. saburreum CCUG 28089(T) confirmed that the bacterium is indeed able to form spores. Based on 16S rRNA gene sequencing, phenotypic and biochemical properties, strains CD3:22(T) and N1(T) represent novel species of a new and distinct genus, named Lachnoanaerobaculum gen. nov., in the family Lachnospiraceae [within the order Clostridiales, class Clostridia, phylum Firmicutes]. Strain CD3:22(T) (=CCUG 58757(T) =DSM 23576(T)) is the type strain of the type species, Lachnoanaerobaculum umeaense gen. nov., sp. nov., of the proposed new genus. Strain N1(T) (=CCUG 60305(T)=DSM 24553(T)) is the type strain of Lachnoanaerobaculum orale sp. nov. Moreover, Eubacterium saburreum is reclassified as Lachnoanaerobaculum saburreum comb. nov. (type strain CCUG 28089(T) =ATCC 33271(T) =CIP 105341(T) =DSM 3986(T) =JCM 11021(T) =VPI 11763(T)).

From its initial discovery in the Gulf of Mexico in the late 1940s until 1990,Perkinsus marinus, the parasite responsible for Dermo disease in the eastern oyster,Crassostrea virginica, was rarely found north of Chesapeake Bay. In 1990-92, an apparent range extension of the parasite led to epizootic outbreaks of the disease over a 500 km range north of Chesapeake Bay. One of the hypotheses for the range extension argues that small, undetected numbers of parasites were already present in northern oysters as the result of repeated historical introductions, and that a sharp warming trend in 1990-92 stimulated the disease outbreak. This argument was based on trends in air temperature. The present study examined this hypothesis by analysing water temperatures, rather than air temperatures, for five stations located in areas affected by the recent epizootics. At all five stations, there was a strong increasing trend in winter sea-surface temperature (SST) between 1986 and 1991. At four of the five stations, there was a smaller increasing trend in winter temperatures after 1960. There were no consistent or obvious trends in summer (August) temperatures. In Delaware Bay, which has a 40 year history of monitoring for oyster diseases, occasional findings ofP. marinusin oysters were correlated with warming episodes that were especially notable in the winter (February) record. Empirical orthogonal function (EOF) analysis showed that winter temperatures varied consistently at the stations examined and were associated with variations inP. marinusprevalence. Associations using EOF analysis with August temperatures were much weaker. The SST record is consistent with the hypothesis that increasing winter water temperatures have been important in the recent outbreak ofP. marinusepizootics in the north-eastern U.S.A.

Birds living in coastal areas are exposed to severe storms and tidal flooding during the nesting season, but also to contaminants that move up the food chain from the water column and sediment to their prey items. We examine metals in Herring Gull (Larus argentatus) and Great Black-backed Gull (Larus marinus) eggs collected from the New York/New Jersey harbor estuary in 2012 and in 2013 to determine if there were significant yearly differences in metal levels. We test the null hypothesis that there were no significant yearly differences in metal levels. We investigate whether there were consistent differences in metals from 2012 to 2013 that might suggest a storm-related effect because Superstorm Sandy landed in New Jersey in October 2012 with high winds and extensive flooding, and view this research as exploratory. Except for arsenic, there were significant inter-year variations in the mean levels for all colonies combined for Herring Gull, and for lead, mercury and selenium for Great Black-backed Gulls. All metal levels in 2013 were less than in 2012, except for lead. These differences were present for individual colonies as well. Metal levels varied significantly among islands for Herring Gulls in both years (except for cadmium in 2013). No one colony had the highest levels of all metals for Herring Gulls. A long term data set on mercury levels in Herring Gulls indicated that the differences between 2012 and 2013 were greater than usual. Several different factors could account for these differences, and these are discussed.

Birds living in coastal areas are exposed to severe storms and tidal flooding during the nesting season, but also to contaminants that move up the food chain from the water column and sediment to their prey items. We examine metals in Herring Gull (Larus argentatus) and Great Black-backed Gull (Larus marinus) eggs collected from the New York/New Jersey harbor estuary in 2012 and in 2013 to determine if there were significant yearly differences in metal levels. We test the null hypothesis that there were no significant yearly differences in metal levels. We investigate whether there were consistent differences in metals from 2012 to 2013 that might suggest a storm-related effect because Superstorm Sandy landed in New Jersey in October 2012 with high winds and extensive flooding, and view this research as exploratory. Except for arsenic, there were significant inter-year variations in the mean levels for all colonies combined for Herring Gull, and for lead, mercury and selenium for Great Black-backed Gulls. All metal levels in 2013 were less than in 2012, except for lead. These differences were present for individual colonies as well. Metal levels varied significantly among islands for Herring Gulls in both years (except for cadmium in 2013). No one colony had the highest levels of all metals for Herring Gulls. A long term data set on mercury levels in Herring Gulls indicated that the differences between 2012 and 2013 were greater than usual. Several different factors could account for these differences, and these are discussed. PMID:25471353

Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, fixes CO2 by the Calvin-Benson-Bassham cycle. Strain MH-110 possesses three different sets of genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO): CbbLS-1 and CbbLS-2, which belong to form I (L8S8), and CbbM, which belongs to form II (Lx). In this paper, we report that the genes for CbbLS-1 (cbbLS-1) and CbbM (cbbM) are both followed by the cbbQO genes and preceded by the cbbR genes encoding LysR-type regulators. In contrast, the gene for CbbLS-2 (cbbLS-2) is followed by genes encoding carboxysome shell peptides. We also characterized the three RubisCOs in vivo by examining their expression profiles in environments with different CO2 availabilities. Immunoblot analyses revealed that when strain MH-110 was cultivated in 15% CO2, only the form II RubisCO, CbbM, was expressed. When strain MH-110 was cultivated in 2% CO2, CbbLS-1 was expressed in addition to CbbM. In the 0.15% CO2 culture, the expression of CbbM decreased and that of CbbLS-1 disappeared, and CbbLS-2 was expressed. In the atmospheric CO2 concentration of approximately 0.03%, all three RubisCOs were expressed. Transcriptional analyses of mRNA by reverse transcription-PCR showed that the regulation was at the transcriptional level. Electron microscopic observation of MH-110 cells revealed the formation of carboxysomes in the 0.15% CO2 concentration. The results obtained here indicate that strain MH-110 adapts well to various CO2 concentrations by using different types of RubisCO enzymes.

This study hypothesizes the existence of three groups of sea lamprey Petromyzon marinus L. in Portugal (North/Central group, Tagus group, and Guadiana group), possibly promoted by seabed topography isolation during the oceanic phase of the life cycle. Within this context, our purpose was to analyze the existence of a stock structure on sea lamprey populations sampled in the major Portuguese river basins using both morphological characters and heart tissue fatty acid signature. In both cases, the multiple discriminant analysis revealed statistically significant differences among groups, and the overall corrected classification rate estimated from cross-validation procedure was particularly high for the cardiac muscle fatty acid profiles (i.e. 83.8%). Morphometric characters were much more useful than meristic ones to discriminate stocks, and the most important variables for group differentiation were eye length, second dorsal fin length and branchial length. Fatty acid analysis showed that all lampreys from the southern Guadiana group were correctly classified and not mixing with individuals from any other group, reflecting a typical heart fatty acid signature. Our results revealed that 89.5% and 72.2% of the individuals from the Tagus and North/Central groups, respectively, were also correctly classified, despite some degree of overlap between individuals from these groups. The fatty acids that contributed to the observed segregation were C16:0; C17:0; C18:1ω9; C20:3ω6 and C22:2ω6. Detected differences are probably related with environmental variables to which lampreys may have been exposed, which leaded to different patterns of gene expression. These results suggest the existence of three different sea lamprey stocks in Portugal, with implication in terms of management and conservation.

Two recently cloned gonadotropin-releasing hormone (GnRH) receptors (lamprey GnRH-R-2 and lamprey GnRH-R-3) along with lamprey (l) GnRH-R-1 were shown to share similar structural features and amino acid motifs common to other vertebrate receptors. Here we report on our findings of RNA expression of these three GnRH receptors in the three major life stages (larval, parasitic, and adult phases) of the sea lamprey, Petromyzon marinus, a basal vertebrate. For each stage, we examined the expression of messenger RNA encoding the receptors in the brain, pituitary, gonad, heart, muscle, liver, eye, intestine, kidney, skin, thyroid, gill, and endostyle by RT-PCR. In adult lampreys, the spatial expression of the three receptors in the brain and pituitary was investigated by in situ hybridization. In general, the receptors were more widely expressed in adult tissues as compared to parasitic-phase tissues and least widely expressed in the larval tissues. There were noted differences in male and female lampreys in the adult and parasitic phases for all three receptors. The data showed the presence of all three receptor transcripts in brain tissues for adult and parasitic phases and all three receptor transcripts were expressed in the adult pituitaries, but not in the parasitic pituitaries. However, in the larval phase, only lGnRH-R-1 was expressed in the larval brain and pituitary. In situ hybridization revealed that lGnRH-R-2 and -3 were expressed in the pineal tissue of adult female lampreys while lGnRH-R-1 was expressed in the pineal in adult male lampreys, all restricted to the pineal pellucida. In summary, these data provide an initial comparative analysis of expression of three lamprey GnRH receptors suggesting differential regulation within males and females at three different life/reproductive stages. PMID:23754972

A Gram-stain-negative, non-motile, non-spore-forming, rod-shaped bacterium, designated strain GYP-24T, was isolated from the culture broth of a marine microalga, Picochlorum sp. 122. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain GYP-24T forms a robust cluster with H.wangdoniaseohaensis KCTC 32177T (95.8 % sequence similarity) in the family Flavobacteriaceae. Growth of strain GYP-24T was observed at 15, 22, 28, 30, 33 and 37 °C (optimal 30-33 °C), pH 6.0-10.0 (optimal pH 7.0-8.0) and in the presence of 0.5-4 % (w/v) NaCl (optimal 2-3 %). The only menaquinone of strain GYP-24T was MK-6, and the G+C content of the genomic DNA was 36.9 mol%. The major fatty acid profile comprised iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω7c/ω6c), iso-C15 : 1 G and iso-C15 : 0. The major polar lipids of strain GYP-24T were phosphatidylethanolamine, one unidentified phospholipid, three unidentified aminolipids and three unidentified lipids. Comprehensive analyses based on polyphasic characterization of GYP-24T indicated that it represents a novel species of a new genus, for which the name Gelatiniphilus marinus gen. nov., sp. nov. is proposed. The type strain is GYP-24T (=KCTC 42903T=MCCC 1K01730T). An emended description of the genus Hwangdonia is also given.

The neuropeptide Y system consists of several neuropeptides acting through a broad number of receptor subtypes, the NPY family of receptors. NPY receptors are divided into three subfamilies (Y1, Y2, and Y5) that display a complex evolutionary history due to local and large-scale gene duplication events and gene losses. Lampreys emerged from a basal branch of the tree of vertebrates and they are in a key position to shed light on the evolutionary history of the NPY system. One member of the Y1 subfamily has been reported in agnathans, but the phylogenetic tree of the Y1 subfamily is not yet clear. We cloned the sequences of the Y1-subtype receptor of Petromyzon marinus and Lampetra fluviatilis to study the expression pattern of this receptor in lampreys by in situ hybridization and to analyze the phylogeny of the Y1-subfamily receptors in vertebrates. The phylogenetic study showed that the Y1 receptor of lampreys is basal to the Y1/6 branch of the Y1-subfamily receptors. In situ hybridization showed that the Y1 receptor is widely expressed throughout the brain of lampreys, with some regions showing numerous positive neurons, as well as the presence of numerous cerebrospinal fluid-contacting cells in the spinal cord. This broad distribution of the lamprey Y1 receptor is more similar to that found in other vertebrates for the Y1 receptor than that of the other members of the Y1 subfamily: Y4, Y8, and Y6 receptors. Both phylogenetic relationship and expression pattern suggest that this receptor is a Y1 receptor.

The sea lamprey (Petromyzon marinus) is a jawless vertebrate at an evolutionary nexus between invertebrates and jawed vertebrates. Lampreys are known to possess the arginine vasotocin (AVT) hormone utilized by all non-mammalian vertebrates. We postulated that the lamprey would possess AVT receptor orthologs of predecessors to the arginine vasopressin (AVP)/oxytocin (OXT) family of G protein-coupled receptors found in mammals, providing insights into the origins of the mammalian V1A, V1B, V2 and OXT receptors. Among the earliest animals to diverge from the vertebrate lineage in which these receptors are characterized is the jawed, cartilaginous elephant shark, which has genes orthologous to all four mammalian receptor types. Therefore, our work was aimed at helping resolve the critical gap concerning the outcomes of hypothesized large-scale (whole-genome) duplication events. We sequenced one partial and four full-length putative lamprey AVT receptor genes and determined their mRNA expression patterns in 15 distinct tissues. Phylogenetically, three of the full-coding genes possess structural characteristics of the V1 clade containing the V1A, V1B and OXT receptors. Another full-length coding gene and the partial sequence are part of the V2 clade and appear to be most closely related to the newly established V2B and V2C receptor subtypes. Our synteny analysis also utilizing the Japanese lamprey (Lethenteron japonicum) genome supports the recent proposal that jawless and jawed vertebrates shared one-round (1R) of WGD as the most likely scenario.

Theory predicts that neutral genetic variation accumulates within populations to a level determined by gains through mutation and losses by genetic drift. This balance results in a characteristic distribution of allelic variation with the maximum allelic difference determined by effective population size. Here, we report a striking departure from these expectations in the form of allelic dimorphism, observed at the majority of seven loci examined in Perkinsus marinus, an important oyster parasite that causes Dermo disease. DNA sequences were collected from five loci flanking microsatellite repeats and two loci coding for superoxide dismutase enzymes that may mediate the parasite's interaction with its host. Based on 474 sequences, sampled across 5000 km of the eastern United States coastline, no more than two alleles were observed at each locus (discounting singletons). Depending on the locus, the common allele ranged in overall frequency from 72% to 92%. At each locus the two alleles differed substantially (3.8% sequence difference, on average), and the among-locus variance in divergences was not sufficient to reject a simultaneous origin for all dimorphisms using approximate Bayesian methods. Dimorphic alleles were estimated to have diverged from a common ancestral allele at least 0.9 million years ago. Across these seven loci, only five other alleles were ever observed, always as singletons and differing from the dimorphic alleles by no more than two nucleotides. Free recombination could potentially have shuffled these dimorphisms into as many as 243 multilocus combinations, but the existence of only ten combinations among all samples strongly supports low recombination frequencies and is consistent with the observed absence of intragenic recombination. We consider several demographic and evolutionary hypotheses to explain these patterns. Few can be conclusively rejected with the present data, but we advance a recent hybridization of ancient divergent lineages

Total mortality rates of lake trout (Salvelinus namaycush) of age VII and older from eastern Lake Superior were estimated from catch curves of age distributions each year in 1968–78. The instantaneous rate of total mortality Z varied from 0.62 to 2.31 in close synchrony with sea lamprey (Petromyzon marinus) wounding rates on lake trout. The regression of transformed Z on the index of lamprey wounding, accounted for over 89% of the variation in lake trout mortality (r2 = 0.893). An iterative method of estimating rates of exploitation u, instantaneous rates of fishing mortality F, K (a constant relating sample catch per unit effort to population size), instantaneous normal natural mortality rate M, and instantaneous rate of mortality due to sea lamprey predationL from the sample catch per unit effort and total catch by the fishery is presented. A second method using the results of a 1970–71 tagging study to estimate the mean F in 1970–77 yielded closely similar results to the above and is presented as corroboration. The estimates of u, F, andM appear to be reasonable. F ranged from 0.17 in 1974 to 0.42 in 1969 and M was estimated at 0.26. L varied from 0.21 in 1974 to 1.70 in 1968. Management implications of various policies concerning sea lamprey control, exploitation, and stocking are discussed.Key words: lake trout, sea lamprey, lamprey control, mortality, predation, Lake Superior, fishery, management

This study hypothesizes the existence of three groups of sea lamprey Petromyzon marinus L. in Portugal (North/Central group, Tagus group, and Guadiana group), possibly promoted by seabed topography isolation during the oceanic phase of the life cycle. Within this context, our purpose was to analyze the existence of a stock structure on sea lamprey populations sampled in the major Portuguese river basins using both morphological characters and heart tissue fatty acid signature. In both cases, the multiple discriminant analysis revealed statistically significant differences among groups, and the overall corrected classification rate estimated from cross-validation procedure was particularly high for the cardiac muscle fatty acid profiles (i.e. 83.8%). Morphometric characters were much more useful than meristic ones to discriminate stocks, and the most important variables for group differentiation were eye length, second dorsal fin length and branchial length. Fatty acid analysis showed that all lampreys from the southern Guadiana group were correctly classified and not mixing with individuals from any other group, reflecting a typical heart fatty acid signature. Our results revealed that 89.5% and 72.2% of the individuals from the Tagus and North/Central groups, respectively, were also correctly classified, despite some degree of overlap between individuals from these groups. The fatty acids that contributed to the observed segregation were C16:0; C17:0; C18:1ω9; C20:3ω6 and C22:2ω6. Detected differences are probably related with environmental variables to which lampreys may have been exposed, which leaded to different patterns of gene expression. These results suggest the existence of three different sea lamprey stocks in Portugal, with implication in terms of management and conservation. PMID:25259723

A Gram-staining-negative bacterial strain (termed CZ-AZ5T) was isolated from a biological filter in a marine recirculating aquaculture system in Tianjin, China. Its taxonomic status was determined using a polyphasic approach. CZ-AZ5T cells were non-spore-forming, non-motile rods, 0.6-0.7 μm wide and 3.0-3.7 μm long. CZ-AZ5T was strictly heterotrophic, aerobic, oxidase-negative, and catalase-positive. Growth occurred in the temperature range 20-40 °C (optimal: 30 °C), pH range 6.0-8.5 (optimal: 7.5), and salinity range 0-5 % (w/v) NaCl (optimal: 1 %). In phylogenetic analyses based on 16S rRNA gene sequences, CZ-AZ5T was assigned to the family Saprospiraceae (phylum Bacteroidetes) and was clustered with the genera Saprospira and Aureispira within the family. It showed highest sequence similarity to Candidatus Haliscomenobacter calcifugiens (86.2 %), followed by Saprospira grandis ATCC 23119T (85.7 %) and Lewinella persica T-3T (85.6 %). DNA G+C content was 40.1 mol %, the major menaquinone was MK-7, and the major cellular fatty acids (>10 %) were C16:1ω7c and iso-C15:0. Our phenotypic, chemotaxonomic, and phylogenetic observations, taken together, led us to conclude that strain CZ-AZ5T represents a new species and genus of the family Saprospiraceae, for which the name Membranicola marinus gen. nov., sp. nov. is proposed. The type strain is CZ-AZ5T (= CGMCC 1.13179T = JCM 18886T).

Eubacterium sp. GLH with Ruminococcus sp. PO1-3 and Clostridium innocuum ES24-06 possessing enzymes involved in the metabolism of glycyrrhizin (GL) was cultured in GAM medium with and without 1.0 mM GL or 1.0 mM glycyrrhetic acid (GA). GL (1.0 mM) enhanced 3alpha-hydroxyglycyrrhetinate (3alpha-hydroxyGA) dehydrogenase activity, GA (1.0 mm) suppressed 3alpha-hydroxyGA dehydrogenase activity, GL beta-D-glucuronidase activity and the mixed bacterial growth, and GL and GA showed almost no change in a lower level of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity during 5 d of culture. GL (1.0 mM) and GA (1.0 mM) were metabolized to a small amount of GA and a negligible amount of 3-oxo-glycyrrhetic acid (3-oxo-GA) and 3alpha-hydroxyGA, and to a negligible amount of 3-oxo-GA, respectively, by these mixed bacteria. These amounts coincided with those of metabolites produced from 1.0 mM GL and 1.0 mM GA added to these mixed bacteria after 24 h culture. Whole bacteria and sonicated bacteria derived from the collection of these mixed bacteria reached a maximal stage and metabolized GL to a relatively large amount of GA and 3-oxo-GA, and a negligible amount of 3alpha-hydroxyGA and GA to a small amount of 3-oxo-GA and 3alpha-hydroxyGA within 180 min. GL beta-D-glucuronidase with 3beta-HSD and 3alpha-hydroxyGA dehydrogenase partially purified from each bacterium was converted GL to 3alpha-hydroxyGA, producing metabolites of about 60% after 10 min of incubation. These mixed bacteria possessed high enzyme activities could produce the metabolites of GL in under one hour under conditions.

The 1960 year class of sea lampreys, Petromyzon marinus, isolated in a tributary of southern Lake Superior continued to yield information on the early life history of the sea lamprey. The larval population persisted and newly metamorphosed individuals were captured from 1966 until the study was terminated in 1972. The average lengths of larvae collected in October (when yearly growth is nearly complete) in successive years from 1966 to 1972 were 111, 113, 112, 114, 121, 128, and 129 mm. The average lengths of transforming lampreys during the same years were 150, 151, 145, 143, 144, 148, and 156 mm. A gradual downstream shift of the population took place. Catches in an inclined-plane trap at the lower end of the study area increased to a peak of 13,244 in the 1968-69 migration year (September 1-August 31), and then steadily decreased. As the number of lampreys decreased in the upper sections and increased in the lower ones, the changes in density were reflected in changes in growth rates. Although the mean length of ammocetes throughout the stream was 111 mm in 1966, it had increased by 1971 to 151 and 143 mm in the upstream sections (IV and V), but to only 115 mm in the densely populated area immediately above the trap. Of a total of 9,889 larvae marked in 1962-68 to study movement and distribution, 2,045 were recovered as larvae and 1,396 as newly transformed adults. Major downstream movements of larvae occurred during high water in April and May, and of transformed lampreys in mid-October through November. Each year about 40% (range, 30-68) of the annual production of transformed lampreys migrated from the Big Garlic River system in one 12-hour period, and 82% by the end of October. The Big Garlic River study proved conclusively that metamorphosis of a single year class occurs over a considerable number of years. Newly metamorphosed individuals were captured in almost steadily increasing numbers from 1965 (age V) to the termination of the study in 1972 (age XII

Nine isolates obtained from a great scallop hatchery in Norway were characterized using a polyphasic approach. Strains were Gram-negative, aerobic and motile rods with oxidative metabolism. Phylogenetic analysis based on the sequences of 16S rRNA and rpoB genes showed that these strains formed two different groups associated with members of the genus Neptuniibacter. DNA-DNA hybridization (DDH) and Average Nucleotide Identity (ANI) demonstrated that the isolates constituted two novel species of this genus, which can be phenotypically differentiated from their closest relatives. The names Neptuniibacter marinus sp. nov. and Neptuniibacter pectenicola sp. nov are proposed, with ATR 1.1(T) (=CECT 8938(T)=DSM 100783(T)) and LFT 1.8(T) (=CECT 8936(T)=DSM 100781(T)) as respective type strains.

We isolated several strains from various clinical samples (five samples of blood, four of intra-abdominal pus and one of infected soft tissue) that were anaerobic, motile or non-motile and Gram-positive rods. Some of the strains formed spores. Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms could be placed within clostridial cluster IV as defined by Collins et al. [(1994). Int J Syst Bacteriol 44, 812-826] and shared more than 99 % sequence similarity with Clostridium orbiscindens DSM 6740(T) and Eubacterium plautii DSM 4000(T). Together, they formed a distinct cluster, with Bacteroides capillosus ATCC 29799(T) branching off from this line of descent with sequence similarities of 97.1-97.4 %. The next nearest neighbours of these organisms were Clostridium viride, Oscillibacter valericigenes, Papillibacter cinnamivorans and Sporobacter termitidis, with sequence similarities to the respective type strains of 93.1-93.4, 91.2-91.4, 89.8-90 and 88.7-89.3 %. On the basis of biochemical properties, phylogenetic position, DNA G+C content and DNA-DNA hybridization, it is proposed to unify Clostridium orbiscindens and Eubacterium plautii in a new genus as Flavonifractor plautii gen. nov., comb. nov., with the type strain Prévot S1(T) (=ATCC 29863(T) =VPI 0310(T) =DSM 4000(T)), and to reassign Bacteroides capillosus to Pseudoflavonifractor capillosus gen. nov., comb. nov., with the type strain CCUG 15402A(T) (=ATCC 29799(T) =VPI R2-29-1(T)).

subgingival flora of diseased sites. The role of any microorganism in the immunopathology of perlo- dontal diseases is based on the ability of the...periodontitis in young adult humans. Infect Inmun 38:1137, 1982. 14. Listgarten, M.: Electron microscopic observations on the bacterial flora of acute

Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermusmarinus, and for novel populations that are related to thermophilic Paenibacilli and an uncultivated subdivision of the little-studied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus, Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify >3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction. PMID:23894306

Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermusmarinus, and for novel populations that are related to thermophilic Paenibacilli and an uncultivated subdivision of the littlestudied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus, Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify .3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction.

Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermusmarinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermusmarinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

The external metamorphosis of the sea lamprey was divided into four stages, based primarily on the condition of the mouth: mouth reduced, mouth fused, mouth enclosed, and mouth elongated. During metamorphosis, the eye enlarged greatly, the snout and mouth region changed from a fleshy hood enclosing a sieve apparatus to a large sucking disc, the nasopore membrane and the branchial area shrank, the branchiopores changed in shape, the general color changed from dark brown and yellow to an intense blue-black dorsally and white ventrally, and the total length increased. Metamorphosis began in early to mid-July and did not take place after August. The duration of external metamorphosis was about 3 months for lampreys transforming under natural conditions. The mean lengths of metamorphosing lampreys from tributaries of lakes Superior and Michigan were 145 and 136 mm, respectively.

Prochlorococcus is a genus of marine cyanobacteria characterized by small cell and genome size, an evolutionary trend toward low GC content, the possession of chlorophyll b, and the absence of phycobilisomes. Whereas many shared derived characters define Prochlorococcus as a clade, many genome-based analyses recover them as paraphyletic, with some low-light adapted Prochlorococcus spp. grouping with marine Synechococcus. Here, we use 18 Prochlorococcus and marine Synechococcus genomes to analyze gene flow within and between these taxa. We introduce embedded quartet scatter plots as a tool to screen for genes whose phylogeny agrees or conflicts with the plurality phylogenetic signal, with accepted taxonomy and naming, with GC content, and with the ecological adaptation to high and low light intensities. We find that most gene families support high-light adapted Prochlorococcus spp. as a monophyletic clade and low-light adapted Prochlorococcus sp. as a paraphyletic group. But we also detect 16 gene families that were transferred between high-light adapted and low-light adapted Prochlorococcus sp. and 495 gene families, including 19 ribosomal proteins, that do not cluster designated Prochlorococcus and Synechococcus strains in the expected manner. To explain the observed data, we propose that frequent gene transfer between marine Synechococcus spp. and low-light adapted Prochlorococcus spp. has created a "highway of gene sharing" (Beiko RG, Harlow TJ, Ragan MA. 2005. Highways of gene sharing in prokaryotes. Proc Natl Acad Sci USA. 102:14332-14337) that tends to erode genus boundaries without erasing the Prochlorococcus-specific ecological adaptations.

Members of the genus Prochlorococcus belong to the most abundant phytoplankton on earth. In contrast to other cyanobacteria, Prochlorococcus is characterized by divinyl-chlorophyll containing light-harvesting complexes and the lack of phycobilisomes. Despite the lack of phycobilisomes, all sequenced genomes of Prochlorococcus possess genes that putatively encode enzymes involved in the biosynthesis of open-chain tetrapyrrole molecules. Here, biochemical evidence is presented indicating that high-light- and low-light-adapted Prochlorococcus ecotypes possess genes encoding functional enzymes for the biosynthesis of open-chain tetrapyrrole molecules. Experiments on recombinant protein as well as through complementation studies of a cyanobacterial insertion mutant revealed the functionality of the bilin reductases investigated.

An infectious agent, which appears to be a virus (RJV) has been isolated from the liver of a wild raccoon which has led to a highly fatal type of disease characterized by conjunctivitis and an elevated serum bilirubin frequently accompanied by jaundice on inoculation of raccoons. Ferrets also appear to be susceptible to infections with this agent.

Prochlorococcus is a genus of marine cyanobacteria characterized by small cell and genome size, an evolutionary trend toward low GC content, the possession of chlorophyll b, and the absence of phycobilisomes. Whereas many shared derived characters define Prochlorococcus as a clade, many genome-based analyses recover them as paraphyletic, with some low-light adapted Prochlorococcus spp. grouping with marine Synechococcus. Here, we use 18 Prochlorococcus and marine Synechococcus genomes to analyze gene flow within and between these taxa. We introduce embedded quartet scatter plots as a tool to screen for genes whose phylogeny agrees or conflicts with the plurality phylogenetic signal, with accepted taxonomy and naming, with GC content, and with the ecological adaptation to high and low light intensities. We find that most gene families support high-light adapted Prochlorococcus spp. as a monophyletic clade and low-light adapted Prochlorococcus sp. as a paraphyletic group. But we also detect 16 gene families that were transferred between high-light adapted and low-light adapted Prochlorococcus sp. and 495 gene families, including 19 ribosomal proteins, that do not cluster designated Prochlorococcus and Synechococcus strains in the expected manner. To explain the observed data, we propose that frequent gene transfer between marine Synechococcus spp. and low-light adapted Prochlorococcus spp. has created a “highway of gene sharing” (Beiko RG, Harlow TJ, Ragan MA. 2005. Highways of gene sharing in prokaryotes. Proc Natl Acad Sci USA. 102:14332–14337) that tends to erode genus boundaries without erasing the Prochlorococcus-specific ecological adaptations. PMID:20333202

Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs. PMID:23293031

Summary The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from E. rectale (ErTadZ), in complex with ATP and Mg2+, was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker-A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down “active site”. Homology modeling of the N-terminal atypical receiver domain of CpaE indicates that it has a conserved protein-protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process. PMID:22211578

We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermusmarinus and show that it conforms to the classical α/β hydrolase fold with an extended ‘lid’ region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries. PMID:27851780

We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermusmarinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-alpha-D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-alpha-D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-alpha-D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.

To compare xylans from forestry with agricultural origins, hardwood xylan (birch) and cereal arabinoxylan (rye) were hydrolyzed using two variants of the xylanase RmXyn10A, full-length enzyme and catalytic module only, from Rhodothermusmarinus . Cultivations of four selected bacterial species, using the xylooligosaccharide (XOS) containing hydrolysates as carbon source, showed selective growth of Lactobacillus brevis DSMZ 1264 and Bifidobacterium adolescentis ATCC 15703. Both strains were confirmed to utilize the XOS fraction (DP 2-5), whereas putative arabinoxylooligosaccharides from the rye arabinoxylan hydrolysate were utilized by only B. adolescentis. Escherichia coli did not grow, despite its capability to grow on the monosaccharides arabinose and xylose. It was also shown that Pediococcus parvulus strain 2.6 utilized neither xylose nor XOS for growth. In summary, RmXyn10A or its catalytic module proved suitable for high-temperature hydrolysis of hardwood xylan and cereal arabinoxylan, producing XOS that could qualify as prebiotics for use in functional food products.

Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60 C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermusmarinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80 C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermusmarinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions. PMID:21724886

The β-barrel assembly machine (BAM) mediates folding and insertion of integral β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Of the five BAM subunits, only BamA and BamD are essential for cell viability. Here we present the crystal structure of a fusion between BamA POTRA4-5 and BamD from Rhodothermusmarinus. The POTRA5 domain binds BamD between its tetratricopeptide repeats 3 and 4. The interface structural elements are conserved in the Escherichia coli proteins, which allowed structure validation by mutagenesis and disulfide crosslinking in E. coli. Furthermore, the interface is consistent with previously reported mutations that impair BamA-BamD binding. The structure serves as a linchpin to generate a BAM model where POTRA domains and BamD form an elongated periplasmic ring adjacent to the membrane with a central cavity approximately 30 × 60 Å wide. We propose that nascent OMPs bind this periplasmic ring prior to insertion and folding by BAM.

The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermusmarinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.

Dihydroorotase (DHOase) catalyzes the formation of dihydroorotate in the de novo pyrimidine biosynthetic pathway. The gene encoding the type I DHOase from the hyperthermophilic bacterium Aquifex aeolicus has been cloned in Escherichia coli with a polyhistidine affinity tag appended to the amino-terminal end and sequenced. The recombinant protein was expressed at high levels and could be purified readily in a single step by Ni(2+) affinity chromatography. Both native and selenomethionine-labeled proteins were crystallized using the hanging-drop vapor-diffusion technique. Screens of the purified protein identified several conditions that yielded crystals; however, the best crystals were obtained using 1 M Li(2)SO(4), 10 mM NiCl(2), 100 mM Tris acetate pH 8.5 as the precipitant. Well formed diamond-shaped crystals appeared within 1 d and continued to grow over several weeks to about 0.5 mm in the largest dimension. The crystals diffract to 1.7 A and belong to space group C2, with unit-cell parameters a = 119.8, b = 88.0, c = 55.2 A, beta = 99.0 degrees and a mosaic spread of 0.6 degrees. There is one DHOase monomer in the asymmetric unit.

The oyster pathogen Perkinsus marinusproduces many extracellular proteins (ECP) in vitro. Analysis of this ECP revealed a battery of hydrolytic enzymes. Some of these enzymes are known to modulate the activity of host defense cells. Although information on the effects of P. marin...

In order to analyze and model the body kinematics used by fish in a wide range of swimming behaviors, we developed a technique to separate the periodic whole-body motions that characterize steady swimming from the secular motions that characterize changes in whole-body shape. We applied this harmonic analysis technique to the study of the forward and backward swimming of lamprey. We found that in order to vary the unsteadiness of swimming, lamprey superimpose periodic and secular components of their body motion, modulate the patterns and magnitudes of those components, and change shape. These kinematic results suggest the following hydromechanical hypothesis: steady swimming is a maneuver that requires active suppression of secular body reconfigurations.

The sex, length, and weight were determined of nearly all recently transformed sea lampreys migrating downstream in the Carp Lake River, Michigan, in the fall, winter, and spring of 1960-61. Similar data were collected from samples of an earlier run in the Carp Lake River and of runs in three other tributaries of Lakes Huron and Michigan. The sex ratio of the 1960-61 migrants in the Carp Lake River was 324 males:100 females. Sex ratios of migrants in the other runs varied from 77 to 86 males:100 females. The high proportion of males in the 1960-61 run in the Carp Lake River is attributed to the effective prevention of recruitment of sea lampreys in the river and transformation of the females at an earlier age than is characteristic of the males. A near equal sex ratio among recently transformed migrants is considered normal for the species. The sex composition of a run changed during the period of migration. The proportion of males among the migrants was greatest at the beginning of the run and declined steadily thereafter. The average size was smaller for males than for females. Differences in the mean lengths and weights of the sexes were statistically significant. The length-weight relation differed for the sexes and showed a slower rate of increase of weight with increase in length than is characteristic of other stages of the animals' life cycle. Seasonal changes in the length-weight relation had a trend toward lower weights among the migrants coming downstream in the later months of the run.

Reproductive functions can be modulated by both stimulatory and inhibitory primer pheromones released by conspecifics. Many stimulatory primer pheromones have been documented, but relatively few inhibitory primer pheromones have been reported in vertebrates. The sea lamprey male sex pheromone system presents an advantageous model to explore the stimulatory and inhibitory primer pheromone functions in vertebrates since several pheromone components have been identified. We hypothesized that a candidate sex pheromone component, 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3 keto-allocholic acid or 3kACA), exerts priming effects through the hypothalamic-pituitary-gonadal (HPG) axis. To test this hypothesis, we measured the peptide concentrations and gene expressions of lamprey gonadotropin releasing hormones (lGnRH) and the HPG output in immature male sea lamprey exposed to waterborne 3kACA. Exposure to waterborne 3kACA altered neuronal activation markers such as jun and jun N-terminal kinase (JNK), and lGnRH mRNA levels in the brain. Waterborne 3kACA also increased lGnRH-III, but not lGnRH-I or -II, in the forebrain. In the plasma, 3kACA exposure decreased all three lGnRH peptide concentrations after 1h exposure. After 2h exposure, 3kACA increased lGnRH-I and -III, but decreased lGnRH-II peptide concentrations in the plasma. Plasma lGnRH peptide concentrations showed differential phasic patterns. Group housing condition appeared to increase the averaged plasma lGnRH levels in male sea lamprey compared to isolated males. Interestingly, 15α-hydroxyprogesterone (15α-P) concentrations decreased after prolonged 3kACA exposure (at least 24h). To our knowledge, this is the only known synthetic vertebrate pheromone component that inhibits steroidogenesis in males.

It has been established that host lipids play a unique role for long term survival and life cycle completion in endogenous parasites. Parasites exploit fatty acids and lipids from the host, not only for membrane synthesis, but also for modification of their surface integrity to a...

In toads, both males and females develop a unique gonadal structure called the Bidder's organ (BO), which resembles ovarian tissue and is attached to the anterior part of the gonad. It is not clear whether the BO is a vestigial organ, or has an endocrine function. In this study, we investigated the expression of the gonadal development genes Dmrt1, Sox9, Sf1, Dax1, and p450arom in the developing BO as compared with the gonads of male and female cane toads. We demonstrate that Sf1, Dax1, and p450arom, key genes involved in vertebrate steroidogenesis, are transcriptionally active in the BO during developmental milestones associated with sexual development and maturation. Furthermore, the pattern of transcriptional activity in the BO is completely independent of the corresponding gonads in both sexes, despite its ovary-like morphology. These results suggest that the BO likely has a steroidogenic role in the development of the cane toad, distinct from that of the gonads.

Reproductive functions can be modulated by both stimulatory and inhibitory primer pheromones released by conspecifics. Many stimulatory primer pheromones have been documented, but relatively few inhibitory primer pheromones have been reported in vertebrates. The sea lamprey male sex pheromone system presents an advantageous model to explore the stimulatory and inhibitory primer pheromone functions in vertebrates since several pheromone components have been identified. We hypothesized that a candidate sex pheromone component, 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3 keto-allocholic acid or 3kACA), exerts priming effects through the hypothalamic-pituitary-gonadal (HPG) axis. To test this hypothesis, we measured the peptide concentrations and gene expressions of lamprey gonadotropin releasing hormones (lGnRH) and the HPG output in immature male sea lamprey exposed to waterborne 3kACA. Exposure to waterborne 3kACA altered neuronal activation markers such as jun and jun N-terminal kinase (JNK), and lGnRH mRNA levels in the brain. Waterborne 3kACA also increased lGnRH-III, but not lGnRH-I or -II, in the forebrain. In the plasma, 3kACA exposure decreased all three lGnRH peptide concentrations after 1 h exposure. After 2 h exposure, 3kACA increased lGnRHI and -III, but decreased lGnRH-II peptide concentrations in the plasma. Plasma lGnRH peptide concentrations showed differential phasic patterns. Group housing condition appeared to increase the averaged plasma lGnRH levels in male sea lamprey compared to isolated males. Interestingly, 15α-hydroxyprogesterone (15α-P) concentrations decreased after prolonged 3kACA exposure (at least 24 h). To our knowledge, this is the only known synthetic vertebrate pheromone component that inhibits steroidogenesis in males.

Although sea lamprey control and heavy plantings of hatchery-reared stock had restored lake trout abundance to prelamprey levels in many areas by 1970, the trout had not yet become self-sustaining. Additional effort will be required to further reduce the effects of lamprey predation.

Sea lampreys from the Lake Huron basin carried no external parasites and showed a fairly low degree of infection by internal parasites. The material examined represented three life-history stages of the sea lamprey. Recently transformed downstream migrants (215 specimens) harbored only nematodes belonging to the genus Camallanus. The percentage of infection was 2.3. Active feeders from the lake (29 lampreys) revealed the highest degree of parasitism (31.0 percent) with the following parasites present: Echinorhynchus coregoni Linkins; Triaenophorus crassus Forel; and Camallanus sp. Among the 257 sexually mature upstream migrants (14.8 percent infected) Echinorhynchus coregoni and E. leidyi Van Cleave were the most common. Only occasional nematodes and cestodes were found, which fact indicates a failure of the lamprey to carry these parasites to the end of its natural life. Of the parasites observed, only the nematodes gave evidence of serious damage to the host. The study suggests that the role played by parasites in the natural control of the sea lamprey in its new habitat in the upper Great Lakes is of minor importance.

The wide-angle optical quality of the cane toad eye was measured using a single-pass intraocular optical fibre microprobe, a double-pass projection method and high magnification ophthalmoscopy to photograph individual photoreceptors. The cane toad eye is a wide angle optical device with a horizontal field of nearly 200 deg and a large relative aperture (ca f/1). Its image quality, which is poor compared to diffraction-limited performance, decreases relatively little towards the periphery. Rough matching was found between image quality and the potential resolution of the rod array, although undersampling occurs for small pupils. Undersampling probably also occurs for the cone and ganglion cell arrays. The relative rotational symmetry of the topographic distribution of image quality precludes direct matching to the topography of the visual streak at the ganglion cell layer although it remains to be determined whether it is matched to the receptor distribution.

The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures. Images PMID:2493632

In higher vertebrates, in response to stress, the hypothalamus produces corticotropin-releasing hormone (CRH), which stimulates cells in the anterior pituitary to produce adrenocorticotropic hormone (ACTH), which in turn stimulates production of either cortisol (F) or corticosterone (B) by the adrenal tissues. In lampreys, however, neither of these steroids is present. Instead, it has been proposed that the stress steroid is actually 17,21-dihydroxypregn-4-ene-3,20-dione (11-deoxycortisol; S). However, there have been no studies yet to determine its mechanism of regulation or site of production. Here we demonstrate that (1) intraperitoneal injections of lamprey-CRH increase plasma S in a dose dependent manner, (2) intraperitoneal injections of four lamprey-specific ACTH peptides at 100μg/kg, did not induce changes in plasma S concentrations in either males or females; (3) two lamprey-specific gonadotropin-releasing hormones (GnRH I and III) and arginine-vasotocin (AVT), all at single doses, stimulated S production as well as, or to an even greater extent than CRH; (4) sea lamprey mesonephric kidneys, in vitro, converted tritiated 17α-hydroxyprogesterone (17α-P) into a steroid that had the same chromatographic properties (on HPLC and TLC) as S; (5) kidney tissues released significantly more immunoassayable S into the incubation medium than gill, liver or gonad tissues. One interpretation of these results is that the corticosteroid production of the sea lamprey, one of the oldest extant vertebrates, is regulated through multiple pathways rather than the classical HPI-axis. However, the responsiveness of this steroid to the GnRH peptides means that a reproductive rather than a stress role for this steroid cannot yet be ruled out.

Anthropogenic environmental stress is a likely contributor to outbreaks of disease due to immunosuppression or increased host vulnerability. Estuarine organisms are exposed to variable concentrations of marine antifouling agents, such as tributyltin (TBT), with higher exposures e...

In higher vertebrates, in response to stress, the hypothalamus produces corticotropin-releasing hormone (CRH), which stimulates cells in the anterior pituitary to produce adrenocorticotropic hormone (ACTH), which in turn stimulates production of either cortisol (F) or corticosterone (B) by the adrenal tissues. In lampreys, however, neither of these steroids is present. Instead, it has been proposed that the stress steroid is actually 17,21-dihydroxypregn-4-ene-3,20-dione (11-deoxycortisol; S). However, there have been no studies yet to determine its mechanism of regulation or site of production. Here we demonstrate that (1) intraperitoneal injections of lamprey-CRH increase plasma S in a dose dependent manner, (2) intraperitoneal injections of four lamprey-specific ACTH peptides at 100 lg/kg, did not induce changes in plasma S concentrations in either males or females; (3) two lamprey-specific gonadotropin-releasing hormones (GnRH I and III) and arginine-vasotocin (AVT), all at single doses, stimulated S production as well as, or to an even greater extent than CRH; (4) sea lamprey mesonephric kidneys, in vitro, converted tritiated 17a-hydroxyprogesterone (17a-P) into a steroid that had the same chromatographic properties (on HPLC and TLC) as S; (5) kidney tissues released significantly more immunoassayable S into the incubation medium than gill, liver or gonad tissues. One interpretation of these results is that the corticosteroid production of the sea lamprey, one of the oldest extant vertebrates, is regulated through multiple pathways rather than the classical HPI-axis. However, the responsiveness of this steroid to the GnRH peptides means that a reproductive rather than a stress role for this steroid cannot yet be ruled out.

This paper reports the identification, expression, binding kinetics, and functional studies of two novel type III lamprey GnRH receptors (lGnRH-R-2 and lGnRH-R-3) in the sea lamprey, a basal vertebrate. These novel GnRH receptors share the structural features and amino acid motifs common to other known gnathostome GnRH receptors. The ligand specificity and activation of intracellular signaling studies showed ligands lGnRH-II and -III induced an inositol phosphate (IP) response at lGnRH-R-2 and lGnRH-R-3, whereas the ligand lGnRH-I did not stimulate an IP response. lGnRH-II was a more potent activator of lGnRH-R-3 than lGnRH-III. Stimulation of lGnRH-R-2 and lGnRH-R-3 testing all three lGnRH ligands did not elicit a cAMP response. lGnRH-R-2 has a higher binding affinity in response to lGnRH-III than lGnRH-II, whereas lGnRH-R-3 has a higher binding affinity in response to lGnRH-II than IGnRH-III. lGnRH-R-2 precursor transcript was detected in a wide variety of tissues including the pituitary whereas lGnRH-R-3 precursor transcript was not as widely expressed and primarily expressed in the brain and eye of male and female lampreys. From our phylogenetic analysis, we propose that lGnRH-R-1 evolved from a common ancestor of all vertebrate GnRH receptors and lGnRH-R-2 and lGnRH-R-3 likely occurred due to a gene duplication within the lamprey lineage. In summary, we propose from our findings of receptor subtypes in the sea lamprey that the evolutionary recruitment of specific pituitary GnRH receptor subtypes for particular physiological functions seen in later evolved vertebrates was an ancestral character that first arose in a basal vertebrate. PMID:22569788

In jawed (gnathostome) vertebrates, the inner ears have three semicircular canals arranged orthogonally in the three Cartesian planes: one horizontal (lateral) and two vertical canals. They function as detectors for angular acceleration in their respective planes. Living jawless craniates, cyclostomes (hagfish and lamprey) and their fossil records seemingly lack a lateral horizontal canal. The jawless vertebrate hagfish inner ear is described as a torus or doughnut, having one vertical canal, and the jawless vertebrate lamprey having two. These observations on the anatomy of the cyclostome (jawless vertebrate) inner ear have been unchallenged for over a century, and the question of how these jawless vertebrates perceive angular acceleration in the yaw (horizontal) planes has remained open. To provide an answer to this open question we reevaluated the anatomy of the inner ear in the lamprey, using stereoscopic dissection and scanning electron microscopy. The present study reveals a novel observation: the lamprey has two horizontal semicircular ducts in each labyrinth. Furthermore, the horizontal ducts in the lamprey, in contrast to those of jawed vertebrates, are located on the medial surface in the labyrinth rather than on the lateral surface. Our data on the lamprey horizontal duct suggest that the appearance of the horizontal canal characteristic of gnathostomes (lateral) and lampreys (medial) are mutually exclusive and indicate a parallel evolution of both systems, one in cyclostomes and one in gnathostome ancestors.

In jawed (gnathostome) vertebrates, the inner ears have three semicircular canals arranged orthogonally in the three Cartesian planes: one horizontal (lateral) and two vertical canals. They function as detectors for angular acceleration in their respective planes. Living jawless craniates, cyclostomes (hagfish and lamprey) and their fossil records seemingly lack a lateral horizontal canal. The jawless vertebrate hagfish inner ear is described as a torus or doughnut, having one vertical canal, and the jawless vertebrate lamprey having two. These observations on the anatomy of the cyclostome (jawless vertebrate) inner ear have been unchallenged for over a century, and the question of how these jawless vertebrates perceive angular acceleration in the yaw (horizontal) planes has remained open. To provide an answer to this open question we reevaluated the anatomy of the inner ear in the lamprey, using stereoscopic dissection and scanning electron microscopy. The present study reveals a novel observation: the lamprey has two horizontal semicircular ducts in each labyrinth. Furthermore, the horizontal ducts in the lamprey, in contrast to those of jawed vertebrates, are located on the medial surface in the labyrinth rather than on the lateral surface. Our data on the lamprey horizontal duct suggest that the appearance of the horizontal canal characteristic of gnathostomes (lateral) and lampreys (medial) are mutually exclusive and indicate a parallel evolution of both systems, one in cyclostomes and one in gnathostome ancestors.

We report here evidence for endogenous NO signalling in long-term (> 1 h) synaptic depression at the neuromuscular junction induced by 20 min of 1 Hz nerve stimulation. Synaptic depression was characterized by a 46% reduction in the end-plate potential (EPP) amplitude and a 21% decrease in miniature EPP (MEPP) frequency, but no change to MEPP amplitude, indicating a reduction in evoked quantal release. Both the membrane-impermeant NO scavenger cPTIO and the NOS inhibitor+ release from the sarcoplasmic reticulum and muscle contraction were blocked with dantrolene. These data suggest that the depression depends on transmission, but not muscle contraction. The calcineurin inhibitors cyclosporin A and FK506, as well as ODQ, an inhibitor of NO-sensitive soluble guanylyl cyclase, Rp-8-pCPT-cGMPS, an inhibitor of cGMP-dependent protein kinase, and the calmodulin antagonist phenoxybenzamine also blocked depression. We propose that low frequency synaptic transmission leads to production of NO at the synapse and depression of transmitter release via a cGMP-dependent mechanism. The NO could be generated either directly from the muscle, or possibly from the Schwann cell in response to an unidentified muscle-derived messenger. We showed that the long-lasting depression of transmitter release was due to sustained activity of the NO signalling pathway, and suggest dephosphorylation of NOS by calcineurin as the basis for continued NO production. PMID:15243135

Endoparasites must breach host barriers to establish infection and then must survive host internal defenses to cause disease. Such barriers may frustrate attempts to experimentally transmit parasites by ?natural' methods. In addition, the host's condition may affect a study's out...

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermusmarinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases

On Helgoland (North Sea), the imagines of Clunio emerge during two seasonal periods (late spring and summer) from water temperatures of 8° 18 °C. The temperature dependence of the known semilunar eclosion rhythm of Clunio (correlated in nature with the spring tides every 14 15 days) was tested in the laboratory. Between 15° and 23 °C the semilunar eclosion maxima varied by only one day within the artifical 15-day zeitgebercycle, below 15 °C they were delayed up to 8 days at 8 °C. However, the days of pupation were approximately independent of the temperature level. One can conclude the existence of a temperature-independent physiological switch inducing the pupation only within a few days of the semilunar zeitgeber-cycle. Moreover, a semilunar synchronized differentiation of the imaginal discs already starts in the preceding larval instar, indicating an additional physiological switch. A model is suggested in which the semilunar eclosion rhythm and its relatively slight temperature dependence is explained by the action of two physiological switches which are coupled with the endogenous temperature-compensated lunar timing mechanism on the same days of the 15-day zeitgeber-cycle. In the laboratory, the diurnal eclosion and its underlying circadian timing mechanism (correlated on Helgoland with the time of spring low water in the late afternoon) also proved to be temperature independent between 12° and 20 °C. A comparison of field and laboratory data showed very similar results at temperatures around 18 °C (summer swarming period). In contrast, the midges emerged on all days of the semimonthly cycle of springs and neaps during the spring swarming period. This lack of semilunar synchronization may be the consequence of fluctuating temperatures during the larval and pupal development in spring time due to a general rise in the water temperature (4° 8 °C) and to short temperature rises up to 18 °C during exposure of the intertidal habitat at about low tide. Since some higher parts of the Clunio habitat suitable for egg deposition are exposed on almost every day of the semimonthly cycle, even such animals that undergo lunar unsynchronized metamorphosis can reproduce within the short imaginal life duration (ca 2 h) if they emerge just about the time of low water. In correspondence with the daily delay in the times of low water by about 50 min, the diurnal eclosion rhythm was in fact modified with the tides during the spring period resulting in shifts of the diurnal eclosion time of up to 12 hours within the semimonthly cycle of springs and neaps.

Diverse mitochondrial (mt) genetic systems have evolved independently of the more uniform nuclear system and often employ modified genetic codes. The organization and genetic system of dinoflagellate mt genomes are particularly unusual and remain an evolutionary enigma. We determined the sequence of full-length cytochrome c oxidase subunit 1 (cox1) mRNA of the earliest diverging dinoflagellate Perkinsus and show that this gene resides in the mt genome. Apparently, this mRNA is not translated in a single reading frame with standard codon usage. Our examination of the nucleotide sequence and three-frame translation of the mRNA suggest that the reading frame must be shifted 10 times, at every AGG and CCC codon, to yield a consensus COX1 protein. We suggest two possible mechanisms for these translational frameshifts: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons or specialized tRNAs recognizing non-triplet codons, AGGY and CCCCU. Regardless of the mechanism, active and efficient machinery would be required to tolerate the frameshifts predicted in Perkinsus mitochondria. To our knowledge, this is the first evidence of translational frameshifts in protist mitochondria and, by far, is the most extensive case in mitochondria. PMID:20507907

Four sea lampreys 345-445 mm long when tagged in November or early December were recovered in mid-September of the following year, at least 2 months after the end of the normal spawning season. Suggested possible reasons for this unusual occurrence are the extension of the parasitic phase of the life cycle beyond the usual 12 to 20 months, late-season maturation of the gonads, disease, or deleterious effects of the tags.

Larval sea lamprey inhabit freshwater streams and migrate to oceans or lakes to feed after a radical metamorphosis; subsequently, mature adults return to streams to spawn. Previous observations suggested that lamprey utilize the odor of conspecific larvae to select streams for spawning. Here we report biochemical and electrophysiological evidence that this odor is comprised of two unique bile acids released by larvae. High performance liquid chromatography and mass spectrometry demonstrated that larval sea lamprey produce and release two unique bile acids, allocholic acid (ACA) and petromyzonol sulfate (PS). Electro-olfactogram (EOG) recording also demonstrated that the olfactory system of migratory adult sea lamprey is acutely and specifically sensitive to ACA and PS; detection thresholds for these compounds were approximately 10(-12) M. ACA and PS were the most potent of 38 bile acids tested and cross-adaptation experiments suggested that adult sea lamprey have specific olfactory receptor sites associated with independent signal transduction pathways for these bile acids. These receptor sites specifically recognize the key substituents of ACA and PS such as a 5 alpha-hydrogen, three axial hydroxyls, and a C-24 sulfate ester or carboxyl. In conclusion, the unique lamprey bile acids, ACA and PS, are potent and specific stimulants of the adult olfactory system, strongly supporting the hypothesis that these unique bile acids function as migratory pheromones in lamprey. PMID:7658193

The first specimen of Ammocoetes branchialis that showed histologically any atrophic changes in the endostyle was taken on July 16. These changes proceeded relatively rapidly for about a month, after which the endostyle as such was no longer recognized. All specimens examined after August 15 showed in cross section the characteristic ductless follicles more or less completely formed. More gradual and minor changes in the way of further absorption of cell remnants and completion of the follicles continued at least until September 1. Two specimens taken from the creek on September 4, 1911. showed complete follicle formation with some stainable colloid (figures 14 and 15). There was still yellow granular pigment in the fibrous tissue between the follicles. In two specimens taken on October 14, 1909, the pigment was absent and the follicles were more closely set, larger, and contained homogenous colloid. In the twenty-four specimens of ammocoetes studied, there were variations in the time of the onset of metamorphosis. There may also be variations in the rate of progress of the changes in different specimens. There is no evidence that removal of the animals from their native environment to the laboratory either increases or decreases the rate of metamorphosis. Schneider states that he was unable to get specimens kept in the laboratory to undergo metamorphosis. Gage, however, has repeatedly observed the metamorphosis under laboratory conditions, and the six of our specimens kept in the laboratory-some for forty days-remained in excellent condition and the metamorphosis proceeded as well as in those living in the creek. I know of no observations bearing on the question as to whether the metamorphosis may be hastened or delayed as it can be in tadpoles and other amphibia. It is probable, however, that physical conditions greatly influence the transformation. These observations as to the length of time from the inception to the completion of metamorphosis indicate that a month and probably longer is necessary for the lake and brook lampreys of Central New York. This is in agreement with the observations of Gage and of Muller on metamorphosis in general, but is at variance with the views of Bujor, who states that the process takes place within three to four days. The first endostylar changes are a gradual shrinkage in the whole organ with thickening of the capsule and septum and proliferation of the connective tissue in the periendostylar zone. The tongue anlage is developed in this thickening just dorsal to the endostyle and anterior to the gland orifice. The size of the chambers progressively decreases and with the thickening of the septum the halves of the endostyle are both absolutely and relatively more separated. All the five types of epithelia are affected, the first to show the change being type I, the four fan-shaped bundles of cuneiform cells of each half of the endostyle. These disappear totally quite early. The next type to show marked changes is type III, or the cells with yellow pigment granules. Here the change is progressive and these cell groups in different stages of atrophy may be traced through to the fully developed follicles. The epithelium of type V, or the endothelial-like lining of the parietal walls of the chambers, is piled up and extruded laterally as the chambers contract or shrink. These cells in different stages of atrophy may be followed until the metamorphosis is nearing completion. It is certain that the cells of types I, III, and V play no part in the formation of the ductless follicles. With types II and IV the question is not so easily settled as it is from one or the other or from both of these types that the permanent follicles arise. One can say definitely that type IV plays the major role, but whether the cells of type II after fusion with the basal group of type IV do not also share in the formation of the ventral follicle of the given chamber, I cannot decide, but from the evidence obtainable this seems probable. It is significant that the cells of type IV are continuous with, and indistinguishable from, the cells lining the orifice and are continued anteriorly in the deep pharyngeal groove and peripharyngeal grooves as well as posteriorally from the orifice in the small pharyngeal groove. As to the fate of this extraglandular epithelium of type IV I have no data save that with the closing of the orifice and the formation of the permanent branchial sac these grooves with their ciliated epithelium disappear and the whole sac comes to be lined with plain stratified epithelium. The fact that the cells of the pharyngeal grooves and the lining cells of the gland orifice are continuous with the cells of the endostyle from which the permanent thyroid follicles are formed is not without significance in relation to the development of the thyroid of the higher chordates. One or more very large follicles are formed from the lower portion of this orificial epithelium of type IV. Four ductless follicles are the maximum number that may be formed primarily in each half of the endostyle from the four areas of epithelium of type IV. From the specimens studied this maximum is frequently not obtained. Posterior to the orifice where four chambers exist, each corresponding to one half of an anterior chamber, but two follicles may be formed from each chamber, but in the coil these are proportionately increased, in cross section. Most of the detailed studies here recorded have been made on the part of the endostyle posterior to the coil where the simplest conditions exist. Here two follicles are ordinarily formed from each chamber. In cross sections the follicles are at first only long tubules whose cavities are the remnants of the original endostyle chambers, but when the metamorphosis is completed each of these primary tubules is cut up into several elongated closed sacs corresponding to the true ductless follicles of all higher chordates. New follicles also arise by budding from these primary ones, and this process is probably of normal occurrence at the metamorphosis.

The toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) appears to be due to a mismatch between ATP supply and demand in lamprey, depleting glycogen stores and starving the nervous system of ATP. The cause of this TFM-induced ATP deficit is unclear. One possibility is that TFM uncouples mitochondrial oxidative phosphorylation, thus impairing ATP production. To test this hypothesis, mitochondria were isolated from the livers of sea lamprey and rainbow trout, and O(2) consumption rates were measured in the presence of TFM or 2,4-dinitrophenol (2,4-DNP), a known uncoupler of oxidative phosphorylation. TFM and 2,4-DNP markedly increased State IV respiration in a dose-dependent fashion, but had no effect on State III respiration, which is consistent with uncoupling of oxidative phosphorylation. To determine how TFM uncoupled oxidative phosphorylation, the mitochondrial transmembrane potential (TMP) was recorded using the mitochondria-specific dye rhodamine 123. Mitochondrial TMP decreased by 22% in sea lamprey, and by 28% in trout following treatment with 50μmolL(-1) TFM. These findings suggest that TFM acted as a protonophore, dissipating the proton motive force needed to drive ATP synthesis. We conclude that the mode of TFM toxicity in sea lamprey and rainbow trout is via uncoupling of oxidative phosphorylation, leading to impaired ATP production.

This study aims to provide a rapid, sensitive and precise UPLC-MS/MS method for target steroid quantitation in biological matrices. We developed and validated an UPLC-MS/MS method to simultaneously determine 16 steroids in plasma and tissue samples. Ionization sources of Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) were compared in this study by testing their spectrometry performances at the same chromatographic conditions, and the ESI source was found up to five times more sensitive than the APCI. Different sample preparation techniques were investigated for an optimal extraction of steroids from the biological matrices. The developed method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99 in broad concentration ranges. The limit of detection (LOD) was from 0.003 to 0.1ng/mL. The method was validated according to FDA guidance and applied to determine steroids in sea lamprey plasma and tissues (fat and testes) by the developed method.

The capture of four recently metamorphosed sea lampreys (two males and two females), 152-172 mm long, in the fall of 1965, established the minimum age at transformation for larvae in the Big Garlic River at 5 years. Age and length (with the exception of a possible minimum length) were determined not to be critical factors in metamorphosis. The presence of larvae 65-176 mm long (mean, 107 mm) in the river in 1965 indicated that metamorphosis of lampreys in a single year class takes place over a period of years.

The NPY receptors known as Y receptors are classified into three subfamilies, Y1, Y2, and Y5, and are involved in different physiological functions. The Y5 receptor is the only member of the Y5 subfamily, and it is present in all vertebrate groups, except for teleosts. Both molecular and pharmacological studies show that Y5 receptor is highly conserved during vertebrate evolution. Furthermore, this receptor is widely expressed in the mammalian brain, including the hypothalamus, where it is thought to take part in feeding and homeostasis regulation. Lampreys belong to the agnathan lineage, and they are thought to have branched out between the two whole-genome duplications that occurred in vertebrates. Therefore, they are in a key position for studies on the evolution of gene families in vertebrates. Here we report the cloning, phylogeny, and brain expression pattern of the sea lamprey Y5 receptor. In phylogenetic studies, the lamprey Y5 receptor clusters in a basal position, together with Y5 receptors of other vertebrates. The mRNA of this receptor is broadly expressed in the lamprey brain, being especially abundant in hypothalamic areas. Its expression pattern is roughly similar to that reported for other vertebrates and parallels the expression pattern of the Y1 receptor subtype previously described by our group, as it occurs in mammals. Altogether, these results confirm that a Y5 receptor is present in lampreys, thus being highly conserved during the evolution of vertebrates, and suggest that it is involved in many brain functions, the only known exception being teleosts.

Seawater acclimation is a critical period for anadromous species and a process yet to be understood in lampreys. Considering that changes in lipid composition of the gill cells' basolateral membranes may disrupt the major transporter Na(+)K(+)-ATPase, the goal of this study was to detect changes at this level during juvenile sea lamprey seawater acclimation. The results showed that saltwater acclimation has a direct effect on the fatty acid composition of gill cells basolateral membrane's phospholipids. When held in full-strength seawater, the fatty acid profile of basolateral membrane's phospholipids suffered a restructure by increasing either saturation or the ratio between oleic acid and eicosapentaenoic acid. Simultaneously, the activity of Na(+)K(+)-ATPase revealed a significant and positive correlation with basolateral membrane's cholesterol content in the presence of highest salinity. Our results pointed out for lipid adjustments involving the functional transporter present on the gill cell basolateral membranes to ensure the role played by branchial Na(+)K(+)-ATPase in ion transport during saltwater acclimation process. The responses observed contributed to the strategy adopted by gill cell's basolateral membranes to compensate for osmotic and ionic stressors, to ensure the success of the process of seawater acclimation associated with the downstream trophic migration of juvenile sea lamprey.

Large biological populations that have not been subdivided should have accumulated variation in different genetic loci. Population samples should identify some loci lacking in variation, others characterized by a few alleles, and still others by many alleles. Departures from these expectations requi...

Recently, two asaccharolytic Eubacterium species, Eubacterium exiguum and Eubacterium lentum, and Peptostreptococcus heliotrinreducens have been reclassified as Slackia exigua, Eggerthella lenta and Slackia heliotrinireducens in the novel genera on the basis of 16S rDNA sequence analysis. But DNA-DNA relatedness among these species and other related bacteria have not been reported yet. DNA-DNA relatedness is the standard arbiter and the recommended method for the designation and evaluation of new species, particularly closely related ones. In the present study, DNA-DNA hybridization studies were performed on S. exigua, S. heliotrinireducens and E. lenta together with the other bacterial species in the related genera. The phylogenetic relationships of these species were also investigated by comparison analysis of 16S rDNA sequence data. In the DNA-DNA hybridization studies, S. exigua showed a DNA homology level of 33% to S. heliotrinireducens and 11% to E. lenta. DNA-DNA homology between S. heliotrinireducens and E. lenta was 10%. But these three species showed very low homology (less than 5%) to the related asaccharolytic species such as Eubacterium and Mogibacterium. In conclusion, the DNA-DNA relatedness data together with the evolutionary data in the present paper further support the reclassification of Eubacterium exiguum, Peptostreptococcus heliotrinreducens and Eubacterium lentum as Slackia exigua, Slackia heliotrinireducens and Eggerthella lenta, respectively.

The influence of freshwater releases and season on disease prevalence and intensity of Perkinsus marinus, condition index, gonadal condition, recruitment potential, and growth of oysters was examined monthly at five locations along the Caloosahatchee estuary, Florida. Temperature...

Influence of water quality and seasonal changes on disease prevalence and intensity of Perkinsus marinus, gonadal condition, recruitment potential, growth of caged juvenile oysters, and habitat suitability of reefs for fishes and macrobenthic invertebrates were measured in Callos...

Perkinsus marinus and Haplosporidium nelsoni cause devasting infections in populations of the eastern oyster, Crassostrea virginica, along the US Atlantic coast and Gulf of Mexico. Salinity and temperature are considered major controlling factors in the prevalence and infection i...

A novel psychrotolerant bacterium strain ISLP-3T was isolated from a sample of naturally formed ice sculpture on the shore of Lake Podprudnoye in Antarctica. Cells were motile, stained Gram positive, non-spore-forming, straight or slightly curved rods with the shape of a baseball bat. The new isolate was facultatively anaerobic and catalase positive. Growth occurred at 3-35 ºC with an optimum at 24 °C, 0-2 % w/v NaCl with an optimum at 0.3 % and pH 6.2 - 9.5 with an optimum 7.5. Strain ISLP-3T grew on several carbon sources, with the best growth on D-cellobiose. The isolate possessed ureolytic activity but growth was inhibited by urea. The strain was sensitive to: ampicillin, gentamycin, kanamycin rifampicin, tetracycline and chloramphenicol. Major fatty acids were: C 15:0 anteiso, C 16:0 iso, C 16:0, C14:0 and C15:0 iso. The predominant menaquinone was MK-9(H4). The genomic G+C content was 69.5 mol%.The 16S rRNA gene possessed 99 % sequence similarity to the gene of Sanguibacter suarezii ST-26T, but their recA genes demonstrated ≤91 % sequence similarity, suggesting that this isolate represented a novel species within genus Sanguibacter. This conclusion was supported by ANI, which was ≤91 % to the most closely related strain. The name Sanguibacter gelidistatuariae sp. nov. is proposed for the new species with the type strain ISLP-3T(= ATCC TSD-17T, = DSM 100501T, = JCM 30887T). Complete genome draft sequence of ISLP-3T was deposited under IMG OID 2657245272. Emendments of related taxa have been made based on experimental data of our comparative analysis.

Background The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. Methodology/Principal Findings The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. Conclusions/Significance While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach. PMID:21283623

Protozoan pathogens represent a serious threat to oyster aquaculture, since they can lead to significant production loses. Moreover, oysters can concentrate human pathogens through filter feeding, thus putting at risk raw oyster consumers' health. Using PCR-based assays in oysters (Crassostrea virginica) from Maine, we expand the Northeast range in the USA for the protozoans Perkinsus marinus, Perkinsus chesapeaki, and Haplosporidium nelsoni, and report for the first time the detection of the human pathogens Toxoplasma gondii and Cryptosporidium parvum. Oysters hosting both P. marinus and P. chesapeaki were more than three times as likely to be infected by a non-Perkinsus than those free of Perkinsus infections.

The known range of the eastern oyster (Crassostrea virginica) parasite, Perkinsus marinus, expanded into the northeastern United States in the early 1990s. We used both in vitro and in vivo data to test the hypothesis that the northward expansion was associated with a low-tempera...

When compared with most vertebrates, frogs use a novel style of jumping locomotion powered by the hindlimbs. Hindlimb bones of frogs must withstand the potentially erratic loads associated with such saltatory locomotion. To evaluate the load bearing capacity of anuran limb bones, we used three-point bending, torsion, and hardness tests to measure the mechanical properties of the femur and tibiofibula from adults of two species that use different jumping styles: explosively jumping bullfrogs (Rana (Lithobates) catesbeiana) and cyclically hopping cane toads (Bufo (Chaunus) marinus). Yield stress and strain values for R. catesbeiana and B. marinus hindlimb bones are within the range of values previously reported for other vertebrates. However, anuran hindlimb bones generally stand out as having higher yield stresses in bending than those of closely related, nonsaltatory salamanders, highlighting the importance of considering phylogenetic context in comparisons of bone functional capacity and adaptation. Stiffness values for both frog species tested were also high, which may facilitate efficient transmission of muscular forces while jumping. Elevated stiffness may also contribute to some discrepancies between determinations of bone properties via hardness versus bending tests. In comparisons between species, B. marinus bones showed significantly higher bending yield stresses than R. catesbeiana, whereas R. catesbeiana bones showed significantly higher torsional yield stresses than B. marinus. These differences may correlate with differences in jumping style and limb anatomy between ranid and bufonid frogs, suggesting that evolutionary changes in bone mechanical properties may help to accommodate new functional demands that emerge in lineages.

Here we report preliminary results from a course of research integrating pathology, feeding ecology, genetics and genomics to address resistance to Dermo disease in eastern oysters. We challenged six oyster families with Perkinsus marinus, the etiological agent of Dermo disease, through either direc...

chemical mechanism for removal of iron from these complexes by cyanobacteria is currently unknown, but genomic experiments should shed useful...dissolved organic matter) are two of the major factors influencing ocean optical properties. The cyanobacteria , including Prochlorocococcus marinus, are...needed for experiments. To complement these studies, analyses of the whole genomic response of Prochlorococcus under iron stress has been undertaken

Juveniles of Lueheia inscripta (Westrumb, 1821 Travassos, 1919 (Acanthocephala: Plagiorhynchidae), an acanthocephalan with six lemnisci, are reported and described from mesenteries of frogs Leptodactylus fragilis Brochi, 1877 and a toad Bufo marinus (Linnaeus, 1758) from Morelos state, Mexico. These are new host records extending the known geographical distribution of this species from Brazil and Puerto Rico to Mexico.

A live in-situ metabolomics capability was developed for prokaryotic cultures under controlled-growth conditions. Toward this goal, a radiofrequency-transparent bioreactor was developed and integrated with a commercial wide-bore nuclear magnetic resonance (NMR) imaging spectrometer and a commercial bioreactor controller. Water suppressed 1H NMR spectroscopy was used to monitor glucose and fructose utilization and byproduct excretion by Eubacterium aggregans (an anaerobic bacterial species relevant for biofuels production) under controlled batch and continuous culture conditions. The resulting metabolite profiles (short chain organic acids and ethanol) and trends are consistent with existing knowledge of its metabolism. However, our study showed the Eubacterium aggregans produces lactate end product in significant concentrations – a result not previously reported. The advantages of live in-situ microbial metabolomics analysis and its complementariness with functional genomics / systems biology methods are discussed.

The main representatives of bacteria in the human colon were investigated by specific PCR and denaturing gradient gel electrophoresis (DGGE). Prevalent in both cases were species of Bifidobacterium, Clostridium, Bacteroides, Faecalibacterium and Eubacterium. Simultaneously, cellulolytic bacteria were isolated from the human feces. The largest proportion was represented by ruminococcus-like isolates. Their presence was confirmed both by PCR and DGGE methods; the latter one was able to give more comprehensive data about the composition of bacterial population in the human colon chyme.

REPORT Carotenoid antenna binding and function in retinal proteins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: Xanthorhodopsin, a proton pump from the...eubacterium Salinibacter ruber, is a unique dual chromophore system that contains, in addition to retinal, the carotenoid salinixanthin as a light... carotenoid ring near the retinal ring. Substitution of the small glycine with bulky tryptophan in this site eliminates binding. The second factor is the 4

Dental implants are commonly used to replace missing teeth. However, the dysbiotic polymicrobial communities of peri-implant sites are responsible for peri-implant diseases, such as peri-implant mucositis and peri-implantitis. In this study, we analyzed the microbial characteristics of oral plaque from peri-implant pockets or sulci of healthy implants (n = 10), peri-implant mucositis (n = 8) and peri-implantitis (n = 6) sites using pyrosequencing of the 16S rRNA gene. An increase in microbial diversity was observed in subgingival sites of ailing implants, compared with healthy implants. Microbial co-occurrence analysis revealed that periodontal pathogens, such as Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, were clustered into modules in the peri-implant mucositis network. Putative pathogens associated with peri-implantitis were present at a moderate relative abundance in peri-implant mucositis, suggesting that peri-implant mucositis an important early transitional phase during the development of peri-implantitis. Furthermore, the relative abundance of Eubacterium was increased at peri-implantitis locations, and co-occurrence analysis revealed that Eubacterium minutum was correlated with Prevotella intermedia in peri-implantitis sites, which suggests the association of Eubacterium with peri-implantitis. This study indicates that periodontal pathogens may play important roles in the shifting of healthy implant status to peri-implant disease. PMID:26077225

Gas chromatography and combined gas chromatography-mass spectrometry have been used to study the fatty acids and hydrocarbons of a bacterium from the Pacific Ocean, Vibrio marinus, a freshwater blue-green alga, Anacystis nidulans, and algal mat communities from the Gulf of Mexico. Both types of microorganisms (bacteria and algae) showed relatively simple hydrocarbon and fatty acid patterns, the hydrocarbons predominating in the region of C-17 and the fatty acids in the range of C-14 to C-18. The patterns of V. marinus were more comparable to those of the algal populations than to patterns reported for other bacteria. An incomplete correlation between fatty acids and hydrocarbons in both types of organisms was observed, making it difficult to accept the concept that the biosynthesis of hydrocarbons follows a simple fatty acid decarboxylation process. PMID:6025301

This study reports on Kudoa spp. (Myxozoa, Multivalvulida) from the fish species Lutjanus analis, Bagre marinus, Aspistor luniscutis and Lutjanus jocu, which were caught in Aracaju, state of Sergipe, Brazil. The parasites formed oval plasmodia around the esophagus of L. analis, and elongated plasmodia inside the skeletal muscle of B. marinus, A. luniscutis and L. jocu. Host myoliquefaction was not observed in all the cases studied. The current study provides a morphological and morphometric description of each parasite as well as a comparison with all the species described worldwide. Lack of molecular data impaired specific identification of the parasites. The importance of these parasites is discussed and the need for further studies on infections in Brazilian fish is emphasized because of the high economic impact of some Kudoa species which cause liquefaction in hosts' muscles and render these fish unsuitable for consumption.

is a long history of using waterborne electric fields in fisheries management (e.g., McMillan 1928), but there are relatively few published...Reynolds. 2002. Electroshock-induced injury in juvenile white sturgeon. North American Journal of Fisheries Management 22: 494-499. Hunn, J. B., and W...D. Youngs. 1980. Role of physical barriers in the control of sea lamprey (Petromyzon marinus). Canadian Journal of Fisheries and Aquatic Sciences 37

Niclosamide to Three Species of Unionid Mussels By Michael A. Boogaard The sea lamprey (Petromyzon marinus), a jawless parasitic eel-like fish native to...to control larval sea lampreys in tribu- taries of the Great Lakes since the early 1960s. The lampricide TFM is the main compound used to keep sea... lamprey populations in check while niclosamide is used primarily in combination with TFM as a cost-sav- ing measure. The addition of niclosamide at

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is used to control sea lamprey (Petromyzon marinus) populations in freshwater lakes. While TFM can have sublethal and lethal effects, little is known about gene expression changes with TFM exposure. Microarray analysis was used to determine differential gene expression over 4 hours of exposure in S. cerevisiae. Among the most significantly up regulated genes were regulators of carbohydrate transport including HXT1, HXT3, HXT4, IMA5, MIG2, and YKR075C. PMID:26606276

THE COAST ARTILLERY JOURNAL Volume 67 OCTOBER, 1927 Number 4 The Beginnings of Coast Fortifications By EDGAR B. WESLEY THE general policy of...the time were withdrawing from l\\ew York to Boston to reinforce the troops there, Marinus Willett, aided by John Morin Scott and others, seized the...Long Island. On September 15, the battalion, as part of General John Morin Scott’s brigade, participated in the retreat from l\\ew York. In October and

The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham’s F12–5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham’s F12–5% FBS– 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham’s F12–5% FBS– 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications. PMID:27149378

The impacts of nonindigenous species on native ecosystems can be severe, sometimes leading to the extinction of native taxa. Interspecific competition is a potential mechanism of negative impact of invasive species, but few studies have conclusively demonstrated competition between native and nonindigenous taxa. In this study I used experimental manipulations to examine the competitive effects of the larvae of two widely introduced anurans, the cane toad, Bufo marinus, and the Cuban treefrog, Osteopilus septentrionalis, on the growth and development of the larvae of two native anurans (the southern toad, Bufo terrestris, and the green treefrog, Hyla cinerea). The presence of O. septentrionalis larvae consistently impacted growth and development of native larvae, resulting in reduced growth rates and delayed metamorphosis of both native species and smaller mass at metamorphosis of B. terrestris. Hyla cinerea larvae transformed at greater body masses when reared with the rapidly transforming nonindigenous species as a result of competitive release. The negative effects of O. septentrionalis on native larvae were generally significant whether native tadpoles were exposed to O. septentrionalis alone or in combination with B. marinus. In contrast, B. marinus tadpoles did not significantly impact the growth or development of either native species. Neither nonindigenous species significantly decreased the survivorship of native larvae, although a trend toward decreased survivorship was evident for H. cinerea. These results suggest that nonindigenous larval anurans may adversely impact native tadpole communities as a result of interspecific competition.

We investigated the nuclear DNA content and genetic diversity of a river lamprey, the Korean lamprey Lampetra morii, which is distributed in the northeast of China. L. morii spends its whole life cycle in fresh water, and its adult size is relatively small (~160 mm long) compared with that of other lampreys. The haploid nuclear DNA content of L. morii is 1.618 pg (approximately 1.582 Gb) in germline cells, and there is ~15% germline DNA loss in somatic cells. These values are significantly smaller than those of Petromyzon marinus, a lamprey with a published draft genome. The chromosomes of L. morii are small and acrocentric, with a diploid modal number of 2n = 132, lower than some other lampreys. Sequence and AFLP analyses suggest that the allelic polymorphism rate (~0.14% based on examined nuclear and mitochondrial DNA sequences) of L. morii is much lower than that (~2%) of P. marinus. Phylogenetic analysis based on a mitochondrial DNA fragment confirms that L. morii belongs to the genus Lampetra, which, together with the genus Lethenteron, forms a sister group to P. marinus. These genetic background data are valuable for subsequent genetic and genomic research on L. morii. PMID:27388621

Animals rely on a mosaic of complex information to find and evaluate mates. Pheromones, often comprised of multiple components, are considered to be particularly important for species-recognition in many species. While the evolution of species-specific pheromone blends is well-described in many insects, very few vertebrate pheromones have been studied in a macro-evolutionary context. Here, we report a phylogenetic comparison of multi-component male odours that guide reproduction in lampreys. Chemical profiling of sexually mature males from eleven species of lamprey, representing six of ten genera and two of three families, indicated the chemical profiles of sexually mature male odours are partially shared among species. Behavioural assays conducted with four species sympatric in the Laurentian Great Lakes indicated asymmetric female responses to heterospecific odours, where Petromyzon marinus were attracted to male odour collected from all species tested but other species generally preferred only the odour of conspecifics. Electro-olfactogram recordings from P. marinusindicated that although P. marinus exhibited behavioural responses to odours from males of all species, at least some of the compounds that elicited olfactory responses were different in conspecific male odours compared to heterospecific male odours. We conclude that some of the compounds released by sexually mature males are shared among species and elicit olfactory and behavioural responses in P. marinus, and suggest that our results provide evidence for partial overlap of male olfactory cues among lampreys. Further characterization of the chemical identities of odour components is needed to confirm shared pheromones among species.

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing.

A marine cyanobacterium, Prochlorococcus, is a unique oxygenic photosynthetic organism, which accumulates divinyl chlorophylls instead of the monovinyl chlorophylls. To investigate the molecular environment of pigments after pigment replacement but before optimization of the protein moiety in photosynthetic organisms, we compared the fluorescence properties of the divinyl Chl a-containing cyanobacteria, Prochlorococcus marinus (CCMP 1986, CCMP 2773 and CCMP 1375), by a Synechocystis sp. PCC 6803 (Synechocystis) mutant in which monovinyl Chl a was replaced with divinyl Chl a. P. marinus showed a single fluorescence band for photosystem (PS) II at 687nm at 77K; this was accompanied with change in pigment, because the Synechocystis mutant showed the identical shift. No fluorescence bands corresponding to the PS II 696-nm component and PS I longer-wavelength component were detected in P. marinus, although the presence of the former was suggested using time-resolved fluorescence spectra. Delayed fluorescence (DF) was detected at approximately 688nm with a lifetime of approximately 29ns. In striking contrast, the Synechocystis mutant showed three fluorescence bands at 687, 696, and 727nm, but suppressed DF. These differences in fluorescence behaviors might not only reflect differences in the molecular structure of pigments but also differences in molecular environments of pigments, including pigment-pigment and/or pigment-protein interactions, in the antenna and electron transfer systems.

The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham's F12-5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham's F12-5% FBS- 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham's F12-5% FBS- 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications.

In this study, the addition of sulfamethazine (SMT) to landfill refuse decreased nitrogen intermediates (e.g. N2O and NO) and dinitrogen (N2) gas fluxes to <0.5 μg-N/kg-refuse·h‑1, while the N2O and N2 flux were at ~1.5 and 5.0 μg-N/kg-refuse·h‑1 respectively in samples to which oxytetracycline (OTC) had been added. The ARG (antibiotic resistance gene) levels in the refuse increased tenfold after long-term exposure to antibiotics, followed by a fourfold increase in the N2 flux, but SMT-amended samples with the largest resistome facilitated the denitrification (the nitrogen accumulated as NO gas at ~6 μg-N/kg-refuse·h‑1) to a lesser extent than OTC-amended samples. Further, deep sequencing results show that long-term OTC exposure partially substituted Hyphomicrobium, Fulvivirga, and Caldilinea (>5%) for the dominant bacterial hosts (Rhodothermus, ~20%) harboring nosZ and norB genes that significantly correlated with nitrogen emission pattern, while sulfamethazine amendment completely reduced the relative abundance of the “original inhabitants” functioning to produce NOx gas reduction. The main ARG carriers (Pseudomonas) that were substantially enriched in the SMT group had lower levels of denitrifying functional genes, which could imply that denitrification is influenced more by bacterial dynamics than by abundance of ARGs under antibiotic pressures.

In this study, the addition of sulfamethazine (SMT) to landfill refuse decreased nitrogen intermediates (e.g. N2O and NO) and dinitrogen (N2) gas fluxes to <0.5 μg-N/kg-refuse·h−1, while the N2O and N2 flux were at ~1.5 and 5.0 μg-N/kg-refuse·h−1 respectively in samples to which oxytetracycline (OTC) had been added. The ARG (antibiotic resistance gene) levels in the refuse increased tenfold after long-term exposure to antibiotics, followed by a fourfold increase in the N2 flux, but SMT-amended samples with the largest resistome facilitated the denitrification (the nitrogen accumulated as NO gas at ~6 μg-N/kg-refuse·h−1) to a lesser extent than OTC-amended samples. Further, deep sequencing results show that long-term OTC exposure partially substituted Hyphomicrobium, Fulvivirga, and Caldilinea (>5%) for the dominant bacterial hosts (Rhodothermus, ~20%) harboring nosZ and norB genes that significantly correlated with nitrogen emission pattern, while sulfamethazine amendment completely reduced the relative abundance of the “original inhabitants” functioning to produce NOx gas reduction. The main ARG carriers (Pseudomonas) that were substantially enriched in the SMT group had lower levels of denitrifying functional genes, which could imply that denitrification is influenced more by bacterial dynamics than by abundance of ARGs under antibiotic pressures. PMID:28120869

Mutant strains of alkalophilic Bacillus firms OF4 that were selected for resistance to duramycin had greatly reduced levels of membrane diacylphosphatidylethanolamine, as had been found in studies of such mutants of Bacillus subtilis. In the B. firmus strains, however, substantial levels of plasmenylethanolamine were found. This is an unusual membrane component for an aerobic eubacterium, but the presence of trace amounts even in the wild type was confirmed in experiments with {sup 32P}{sub i}-labeled growth medium. The membrane lipid composition of the duramycin-resistant strains had several other changes that also left alkalophilic growth unimpaired.

A long-chain fatty acid degrading (beta oxidizing), obligate proton-reducing, acetogenic bacterium strain SD2 of the genus Syntrophomonas has been isolated in coculture with a hydrogen-using bacterium, Desulfovibrio strain G-11. The enzymology of fatty acid degradation is being studied to discover the differences of SD2 from S. wolfei which allow it to degrade long chain fatty acids. A new species, Clostridium pfennigii (V5-2) was isolated from the rumen. A new genus and species, Syntrophococcus sucromutans (S195) is present in relatively high numbers in rumen contents. Another new species is Eubacterium oxidoreducens. (ACR)

The outer surface of the murein sacculus of the eubacterium Bacillus alvei is covered by a surface layer (S-layer) glycoprotein. The glycan chain of this S-layer glycoprotein consists of trisaccharide repeating units with ManNAc, Gal, and Glc as constituents. From cell extracts of B. alvei, nucleotide-activated derivatives of ManNAc, Gal, Glc, and GlcNAc were isolated. Furthermore, GDP- and dolichyl-activated oligosaccharides were obtained. On the basis of the isolated putative glycoprotein precursors, a pathway for the biosynthesis of the oligosaccharide chain is proposed. PMID:8331079

We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes. A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium. The archaebacterium, a thermoacidophil resembling extant Thermoplasma, generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur. Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium “Thiodendron latens.” By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved. This “earliest branching protist” that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts. The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector. As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei. The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera). This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa. PMID:10860956

Facultatively anaerobic and strictly anaerobic bacteria colonizing the intestinal tracts of 14-day-old chicks fed a corn-based diet were enumerated, isolated, and identified. Colony counts from anaerobic roll tubes (rumen fluid medium) or aerobic plates (brain heart infusion agar) recovered from homogenates of the duodenum, upper and lower ileum, and cecum varied appreciably among samples from individual birds. Anaerobic and aerobic counts from the duodenum and ileum were similar. Anaerobic counts were highest from the cecum (0.7 X 10(11) to 1.6 X 10(11)/g of dry tissue) and exceeded aerobic plate counts by a factor of at least 10(2). Facultatively anaerobic groups (Streptococcus, Staphylococcus, Lactobacillus, and Escherichia coli) comprised the predominant flora of the duodenum and ileum, although large numbers of anaerobes (9 to 39% of the small intestine isolates), represented by species of Eubacterium, Propionibacterium, Clostridium, Gemmiger, and Fusobacterium, were also recovered. Strict anaerobes (anaerobic gram-positive cocci, Eubacterium, Clostridium Gemmiger, Fusobacterium, and Bacteriodes) made up nearly the entire microbial population of the cecum. Scanning electron microscopy of the intestinal epithelia of chicks revealed populations of microbes on the duodenal, ileal, and cecal mucosal surfaces. Images PMID:646359

We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes. A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium. The archaebacterium, a thermoacidophil resembling extant Thermoplasma, generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur. Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium "Thiodendron latens." By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved. This "earliest branching protist" that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts. The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector. As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei. The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera). This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa.

The normal microbiota plays an important role in the health of the host, but little is known of how the human immune system recognizes and responds to Gram-positive indigenous bacteria. We have investigated cytokine responses of peripheral blood mononuclear cells (PBMC) to Gram-positive cell walls (CW) derived from four common intestinal indigenous bacteria, Eubacterium aerofaciens (Eu.a.), Eubacterium limosum(Eu.l.), Lactobacillus casei(L.c.), and Lactobacillus fermentum (L.f.). Our results indicate that Gram-positive CW of the normal intestinal microbiota can induce cytokine responses of the human PBMC. The profile, level and kinetics of these responses are similar to those induced by lipopolysaccharide (LPS) or CW derived from a pathogen, Streptococcus pyogenes (S.p.). Bacterial CW are capable of inducing production of a proinflammatory cytokine, tumour necrosis factor-alpha (TNF-α), and an anti-inflammatory cytokine, IL-10, but not that of IL-4 or interferon-gamma (IFN-γ). Monocytes are the main cell population in PBMC to produce TNF-α and IL-10. Induction of cytokine secretion is serum-dependent; both CD14-dependent and -independent pathways are involved. These findings suggest that the human cytokine responses induced by Gram-positive CW of the normal intestinal microbiota are similar to those induced by LPS or Gram-positive CW of the pathogens. PMID:10540188

Butyrate-producing bacteria play an important role in the human colon, supplying energy to the gut epithelium and regulating host cell responses. In order to explore the diversity and culturability of this functional group, we designed degenerate primers to amplify butyryl-CoA:acetate CoA-transferase sequences from faecal samples provided by 10 healthy volunteers. Eighty-eight per cent of amplified sequences showed >98% DNA sequence identity to CoA-transferases from cultured butyrate-producing bacteria, and these fell into 12 operational taxonomic units (OTUs). The four most prevalent OTUs corresponded to Eubacterium rectale, Roseburia faecis, Eubacterium hallii and an unnamed cultured species SS2/1. The remaining 12% of sequences, however, belonged to 20 OTUs that are assumed to come from uncultured butyrate-producing strains. Samples taken after ingestion of inulin showed significant (P=0.019) increases in Faecalibacterium prausnitzii. Because several of the dominant butyrate producers differ in their DNA % G+C content, analysis of thermal melt curves obtained for PCR amplicons of the butyryl-CoA:acetate CoA-transferase gene provides a convenient and rapid qualitative assessment of the major butyrate producing groups present in a given sample. This type of analysis therefore provides an excellent source of information on functionally important groups within the colonic microbial community.

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

Soil, water, and amphibian tissues collected between 1995 and 1999 from 15 study sites in Bermuda were analysed for pesticides and heavy metals. The most abundant pesticide residue in soil was p,p'-dichlorodiphenyldichloroethylene (DDE) which was found at all sites in concentrations ranging from 0.003 to 4.023 p.p.m. No pesticide residues were found in water. DDE was also recovered from the livers and fat bodies of marine toads (Bufo marinus) and whistling frogs (Eleutherodactylus johnstonei). Analyses of food sources consumed by these anuran species revealed residue levels of p, p'-DDE ranging from 0.05 to 0.217 p.p.m. Other soil residues included dichlorodiphenyltrichloroethane (DDT) at eight study sites, Dicofol(kelthane) at eight sites, dieldrin at five sites, and polychlorinated biphenyls (PCBs) as Arochlor 1254 and Arochlor 1260 at seven sites. Analyses of toad livers revealed significant concentrations of cadmium, chromium, copper and zinc. Livers of Bermuda toads exhibited altered hepatocytic morphology and an increased number of melanomacrophages and possible granulomas, while spleens showed a marked decrease in white pulp. Spleen cells from Bufo marinus collected at one site having high levels of cadmium exhibited a decreased B cell response to lipopolysaccharide. The incidence of trematode infection in Bufo marinus increased from 53.8% in 1995 to 90% in 1999. Deformity rates in the limbs of subadult and adult toads ranged between 15 and 25%. Examination of 1,995 newly-metamorphosed toads revealed deformity rates as high as 47%. The current comprehensive study suggests that environmental pollutants may account for immunosuppression, increased susceptibility to infections, limb malformations and possible decline in amphibian populations from Bermuda.

Regions of specialization for water absorption across the skin of Bufonid and Ranid anurans were identified by immunohistochemistry and Western blot analysis, using antibodies raised against arginine vasotocin (AVT)-stimulated aquaporins (AQPs) that are specific to absorbing regions of Hyla japonica. In Bufo marinus, labeling for Hyla urinary bladder-type AQP (AQP-h2), which is also localized in the urinary bladder, occurred in the ventral surface of the hindlimb, pelvic, and pectoral regions. AQP-h2 was not detected in any skin regions of Rana catesbeiana, Rana japonica, or Rana nigromaculata. Hyla ventral skin-type AQP (AQP-h3), which is found in the ventral skin but not the bladder of H. japonica, was localized in the hindlimb, pelvic, and pectoral skins of Bufo marinus, in addition to AQP-h2. AQP-h3 was also localized in ventral skin of the hindlimb of all three Rana species and also in the pelvic region of R. catesbiana. Messenger RNA for AQP-x3, a homolog of AQP-h3, could be identified by RT-PCR from the hindlimb, pectoral, and pelvic regions of the ventral skin of Xenopus laevis, although AVT had no effect on water permeability. In contrast, 10(-8) M AVT-stimulated water permeability and translocation of AQP-h2 and AQP-h3 into the apical membrane of epithelial cells in regions of the skin of species where they had been localized by immunohistochemistry and Western blot analysis. Finally, water permeability of the hindlimb skin of B. marinus and all the Rana species was stimulated by hydrins 1 and 2 to a similar level as seen for AVT. The present data demonstrate species differences in the occurrence, distribution, and regulation of AQPs in regions of skin specialized for rapid water absorption that can be associated with habitat and also phylogeny.

Monogalactosyldiacylglycerol (MGDG) is ubiquitous in the photosynthetic membranes of cyanobacteria and chloroplasts. It is synthesized by galactosylation of diacylglycerol (DAG) in the chloroplasts, whereas it is produced by epimerization of monoglucosyldiacylglycerol (GlcDG) in at least several cyanobacteria that have been analyzed such as Synechocystis sp. PCC 6803. A previous study, however, showed that the mgdE gene encoding the epimerase is absent in some cyanobacteria such as Gloeobacter violaceus, Thermosynechococcus elongatus and Acaryochloris marina. In addition, the N-terminal 'fatty acid hydroxylase' domain is lacking in the MgdE protein of Prochlorococcus marinus. These problems may cast doubt upon the general (or exclusive) role of MgdE in the epimerization of GlcDG to MGDG in cyanobacteria. In addition, GlcDG is usually present at a very low level, and the structural determination of endogenous GlcDG has not been accomplished with cyanobacterial samples. In this study, I determined the structure of GlcDG from Anabaena variabilis by (1)H- and (13)C-nuclear magnetic resonance (NMR) spectroscopy. I then showed that G. violaceus, T. elongatus, A. marina and P. marinus contain GlcDG. In all cases, GlcDG consisted of fewer unsaturated molecular species than MGDG, providing further evidence that GlcDG is a precursor to MGDG. The conversion of GlcDG to MGDG was also demonstrated by radiolabeling and chase experiments in G. violaceus and P. marinus. These results demonstrate that all the analyzed cyanobacteria contain GlcDG, which is converted to MGDG, and suggest that an alternative epimerase is required for MGDG synthesis in these cyanobacteria.

Diel-cycling hypoxia is widespread in shallow portions of estuaries and lagoons, especially in systems with high nutrient loads resulting from human activities. Far less is known about the effects of this form of hypoxia than deeper-water seasonal or persistent low dissolved oxygen. We examined field patterns of diel-cycling hypoxia and used field and laboratory experiments to test its effects on acquisition and progression of Perkinsus marinus infections in the eastern oyster, Crassostrea virginica, as well as on oyster growth and filtration. P. marinus infections cause the disease known as Dermo, have been responsible for declines in oyster populations, and have limited success of oyster restoration efforts. The severity of diel-cycling hypoxia varied among shallow monitored sites in Chesapeake Bay, and average daily minimum dissolved oxygen was positively correlated with average daily minimum pH. In both field and laboratory experiments, diel-cycling hypoxia increased acquisition and progression of infections, with stronger results found for younger (1-year-old) than older (2-3-year-old) oysters, and more pronounced effects on both infections and growth found in the field than in the laboratory. Filtration by oysters was reduced during brief periods of exposure to severe hypoxia. This should have reduced exposure to waterborne P. marinus, and contributed to the negative relationship found between hypoxia frequency and oyster growth. Negative effects of hypoxia on the host immune response is, therefore, the likely mechanism leading to elevated infections in oysters exposed to hypoxia relative to control treatments. Because there is considerable spatial variation in the frequency and severity of hypoxia, diel-cycling hypoxia may contribute to landscape-level spatial variation in disease dynamics within and among estuarine systems. PMID:25671595

The deep chlorophyll maximum (DCM) is a zone of maximal photosynthetic activity, generally located toward the base of the photic zone in lakes and oceans. In the tropical waters, this is a permanent feature, but in the Mediterranean and other temperate waters, the DCM is a seasonal phenomenon. The metagenome from a single sample of a mature Mediterranean DCM community has been 454 pyrosequenced both directly and after cloning in fosmids. This study is the first to be carried out at this sequencing depth (ca. 600 Mb combining direct and fosmid sequencing) at any DCM. Our results indicate a microbial community massively dominated by the high-light-adapted Prochlorococcus marinus subsp. pastoris, Synechococcus sp., and the heterotroph Candidatus Pelagibacter. The sequences retrieved were remarkably similar to the existing genome of P. marinus subsp. pastoris with a nucleotide identity over 98%. Besides, we found a large number of cyanophages that could prey on this microbe, although sequence conservation was much lower. The high abundance of phage sequences in the cellular size fraction indicated a remarkably high proportion of cells suffering phage lytic attack. In addition, several fosmids clearly belonging to Group II Euryarchaeota were retrieved and recruited many fragments from the total direct DNA sequencing suggesting that this group might be quite abundant in this habitat. The comparison between the direct and fosmids sequencing revealed a bias in the fosmid libraries against low-GC DNA and specifically against the two most dominant members of the community, Candidatus Pelagibacter and P. marinus subsp. pastoris, thus unexpectedly providing a feasible method to obtain large genomic fragments from other less prevalent members of this community.

The present study has characterized gonadotropic releasing hormone (GnRH)-like molecules in the brains of representatives of the two southern hemisphere families of lampreys, Geotriidae and Mordaciidae. Chromatographic and immunocytochemical evidence showed that the brains of Geotria australis and Mordacia mordax contain two forms of GnRH-like molecules. These two forms correspond to lamprey GnRH-I and -III, which were first sequenced from the brain of the anadromous sea lamprey Petromyzon marinus, a representative of the family Petromyzontidae that is found only in the northern hemisphere. In chromatographic studies (HPLC) using lamprey GnRH-I and -III antiserum, two early eluting GnRH forms coeluted with synthetic lamprey GnRH-I and -III standards. Our studies thus indicate that, despite their apparently long period of separation, the three families of extant lampreys have each retained both of the lamprey GnRH (-I and -III forms) molecules. Moreover, immunocytochemical localization of lamprey GnRH indicated that the pattern of its distribution in the adult brain of at least one of these southern hemisphere lampreys (G. australis) is similar to that previously described for P. marinus. Distribution of GnRH in the brain of larval G. australis was not as extensive as that in larval P. marinus, which may account for the later gonadal development in the former species. The fact that lamprey GnRH-I and -III are the dominant GnRH forms in all three families of lampreys implies that these neurohormones have an ancient origin.

The effects of serotonin altering parasites, serotonin, the anti-depressant fluoxetine, plus two other highly prescribed pharmaceuticals (carbamazepine and diclofenac) on the behaviour of the marine amphipod, Echinogammarus marinus were investigated. Acanthocephalan parasites are known to alter the swimming behaviour in their amphipod hosts through changes in serotonergic activity resulting in increased predation. Behavioural assays were adapted to record changes in phototaxis and geotaxis behaviour in male E. marinus following 7, 14 and 21 days exposure to serotonin and each pharmaceutical compound at 4 concentrations compared to a control (between 10 ng/L and 10 microg/L). E. marinus infected with acanthocephalans parasites had both significantly higher phototaxis and geotaxis scores than those of uninfected specimens. Phototaxis and geotaxis behaviour increased significantly in a concentration-dependent manner with exposure to serotonin. Fluoxetine significantly altered phototaxis and geotaxis activity in what appeared to be a non-monotonic concentration response curve with the greatest behavioural changes observed at 100 ng/L. The main patterns of these behavioural responses were consistent between two trials and the 3 weeks exposure with specimens spending more time within the light and occurring higher in the water column. No obvious trends could be concluded in the phototaxis and geotaxis scores from individuals exposed to carbamazepine or diclofenac as might be expected from their known mode of action. From this study phototaxis and geotaxis behaviour have been observed to be affected by exposure to serotonin modulators. Parasite studies have shown strong links between changes in behaviour and increased predation risk correlating with changes in serotonergic activity. This study has highlighted the potential for highly prescribed anti-depressant drugs to change the behaviour of an ecologically relevant marine species in ways which could conceivably lead to

Muscles power movement, yet the conceptual link between muscle performance and locomotor performance is poorly developed. Frog jumping provides an ideal system to probe the relationship between muscle capacity and locomotor performance, because a jump is a single discrete event and mechanical power output is a critical determinant of jump distance. We tested the hypothesis that interspecific variation in jump performance could be explained by variability in available muscle power. We used force plate ergometry to measure power produced during jumping in Cuban tree frogs (Osteopilus septentrionalis), leopard frogs (Rana pipiens) and cane toads (Bufo marinus). We also measured peak isotonic power output in isolated plantaris muscles for each species. As expected, jump performance varied widely. Osteopilus septentrionalis developed peak power outputs of 1047.0 ± 119.7 W kg−1 hindlimb muscle mass, about five times that of B. marinus (198.5 ± 54.5 W kg−1). Values for R. pipiens were intermediate (543.9 ± 96.2 W kg−1). These differences in jump power were not matched by differences in available muscle power, which were 312.7 ± 28.9, 321.8 ± 48.5 and 262.8 ± 23.2 W kg−1 muscle mass for O. septentrionalis, R. pipiens and B. marinus, respectively. The lack of correlation between available muscle power and jump power suggests that non-muscular mechanisms (e.g. elastic energy storage) can obscure the link between muscle mechanical performance and locomotor performance. PMID:21502120

Predation by raccoons, Procyon lotor marinus (L.), is the primary cause of sea turtle nest loss in the Ten Thousand Islands archipelago. Four islands within Ten Thousand Islands National Wildlife Refuge were surveyed for sea turtle nesting activity from 1991-95. Raccoons depredated 76-100% of nests on Panther Key from 1991-94, until 14 raccoons were removed in 1995 resulting in 0% depredation and 2 more were removed in 1996 resulting in 0% depredation. Raccoon removal may be an effective management option for increasing sea turtle nest survival on barrier islands.

Bayluscide (5,2'-dichloro-4'-nitrosalicylanilide) and TFM (3-trifluoromethyl-4-nitrophenol) were evaluated as selective larvicides for control of the sea lamprey, Petromyzon marinus, in the Big Garlic River and Saux Head Lake in Marquette County, Michigan. Population estimates and movement of ammocetes were determined from the recapture of marked ammocetes released before chemical treatment. In 1966 the estimated population of 3136 ammocetes off the stream mouth in Saux Head Lake was reduced 89% by treatment with granular Bayluscide; this percentage was supported by a population estimate of 120 ammocetes in 1967, an indicated reduction of 96% from 1966. Post-marking movement of ammocetes was greater upstream than downstream.

Ergasilus megaceros (Copepoda: Ergasilidae) was recovered from the nasal fossae (lamellae) of the olfactory sac in 1 (1.8%) of 56 sea lampreys, Petromyzon marinus Linne, 1758, collected in May 2002 from the Cheboygan River, Michigan. Although the sea lamprey is a new host record for E. megaceros, this fish species may not be a preferred host because of its low prevalence. Ergasilus megaceros is the second ergasilid species reported from the sea lamprey in North America. This is the third report of an ergasilid species infecting the nasal fossae of fishes in North America, with E. rhinos being the only other species reported from this site.

The widespread decline of Pacific lamprey (Lampetra tridentata) in the Pacific Northwest, especially in the Columbia River system has led to concerns and questions from a number of regional agencies, Native American tribes, and the public. To address these concerns, new research efforts must focus on specific problems associated with this understudied species. The preservation and restoration of this species is critical for a number of reasons, including its importance to the tribes and its importance as an indicator of ecosystem health. Historically lamprey have been labeled a pest species due to the problems associated with the exotic sea lamprey, (Petromyzon marinus), invading the Great Lakes.

Abstract A taxonomic study of the South Korean Hydraenidae is presented. Eight species in two genera are recognized, one of which is reported for the first time in the Korean peninsula, Ochthebius marinus (Paykull). It was also found that Hydraena riparia Kugelann and Ochthebius inermis Sharp previously recorded in South Korea were incorrect identifications of Hydraena puetzi Jäch and Ochthebius lobatus Pu, respectively. Habitus and SEM photographs, line drawings of aedeagus, distribution maps, keys, and redescriptions of the species are provided. PMID:27917050

The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.

Lampreys are eel-like jawless fishes evolutionarily positioned between invertebrates and vertebrates, and have been used as model organisms to explore vertebrate evolution. In this study we determined the complete genome sequence of the mitochondrial DNA of the Japanese river lamprey, Lethenteron japonicum, using next-generation sequencers. The sequence was 16,272 bp in length. The gene content and order were identical to those of the sea lamprey, Petromyzon marinus, which has been the reference among lamprey species. However, the sequence similarity was less than 90%, suggesting the need for the whole-genome sequencing of L. japonicum.

A numerical taxonomic study was performed on 65 Gram-positive wild strains of heterotrophic, aerobic, marine bacteria, and 9 reference strains. The isolates were obtained from oysters and seawater sampled monthly over one year, by direct plating on Marine Agar. The strains were characterized by 96 morphological, biochemical, physiological and nutritional tests. Clustering yielded 13 phena at 0.62 similarity level (Sl coefficient). Only one of the seven phena containing wild isolates could be identified (Bacillus marinus). A pronounced salt requirement was found in most isolates.

This review assesses and examines the work conducted to date concerning host and parasite interactions between marine bivalve molluscs and protozoan parasites, belonging to Perkinsus species. The review focuses on two well-studied host-parasite interaction models: the two clam species, Ruditapes philippinarum and R. decussatus, and the parasite Perkinsus olseni, and the eastern oyster, Crassostrea virginica, and the parasite Perkinsus marinus. Cellular and humoral defense responses of the host in combating parasitic infection, the mechanisms (e.g., antioxidant enzymes, extracellular products) employed by the parasite in evading host defenses as well as the role of environmental factors in modulating the host-parasite interactions are described.

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) has been used in the United States and Canada for more than 30 years to control populations of sea lamprey (Petromyzon marinus) in the Great Lakes. There is concern that sea lamprey might become resistant to TFM. Lampricide toxicity tests have been conducted at the Hammond Bay Biological Station, Millersburg, Michigan, since the 1950s and examination of TFM toxicity data for larval lamprey from 1963 to 1987 indicated that sea lamprey have not developed increased resistance to TFM. Maintenance of current control practices are unlikely to cause the development of TFM-resistant sea lamprey strains in the foreseeable future.

Colorimetric methods are described for the detection of residues of the selective lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), in natural waters and fish tissues. Solvent extraction and anion exchange were used to separate and concentrate the lampricide, and to reduce high background colors which often interfere with the determinations. Data from the Pentwater and Muskegon Rivers (Michigan) showed an exponential rate of disappearance of TFM from the water immediately after treatments to eradicate larvae of the sea lamprey (Petromyzon marinus). No TFM was recovered from fish exposed in their natural environment to low levels of the lampricide during treatment; residues were recovered, however, from fish exposed to high concentrations in the laboratory.

A radiative transfer computer model was developed to characterize the total flux of chlorophyll a fluoresced or backscattered photons when laser radiation is incident on turbid water that contains a non-homogeneous suspension of inorganic sediments and phytoplankton. The radiative transfer model is based on the Monte Carlo technique and assumes that: (1) the aquatic medium can be represented by a stratified concentration profile; and (2) that appropriate optical parameters can be defined for each layer. The model was designed to minimize the required computer resources and run time. Results are presented for an anacystis marinus culture.

AN INVESTIGATION OF THE LARVAL STAGES OF THE SEA LAMPREY (Petromyzon marinus), which started in 1960, required a trap that could capture lamprey larvae moving downstream, and that would operate efficiently throughout the year. Sub zero temperatures in the winter and flooding during the spring were the most critical operational problems. Part of the trap was based on the principles of an inclined-plane trap described by Wolf (1951), but the design also incorporated features to prevent ice formation during the winter and to and to strain high flows during the spring runoff.

We describe a water-recirculating system for inducing spawning of sea lampreys (Petromyzon marinus) held under laboratory conditions. Water temperature in the system was gradually increased to and maintained at 18 +/- 2 degrees C, the optimal temperature for spawning. About 10% freshwater was added daily to prevent buildup of waste products. Sea lampreys were provided substrate (approximately 3-6 cm in diameter) to build nests, and a water velocity of 0.2-0.3 m/s was maintained with an electric trolling motor. Sea lampreys held in this system exhibited characteristic spawning behavior. Prolarvae produced from artificial fertilization of gametes developed according to the standard timeline.

A bacterial strain, designated RA, was isolated from water sample of a hot spring on Langkawi Island of Malaysia using marine agar. Strain RA is an aerophilic and thermophilic microorganism that grows optimally at 50–60°C and is capable of growing in marine broth containing 1–10% (w/v) NaCl. 16S rRNA gene sequence analysis demonstrated that this strain is most closely related (<90% sequence identity) to Rhodothermaceae, which currently comprises of six genera: Rhodothermus (two species), Salinibacter (three species), Salisaeta (one species), Rubricoccus (one species), Rubrivirga (one species), and Longimonas (one species). Notably, analysis of average nucleotide identity (ANI) values indicated that strain RA may represent the first member of a novel genus of Rhodothermaceae. The draft genome of strain RA is 4,616,094 bp with 3630 protein-coding gene sequences. Its GC content is 68.3%, which is higher than that of most other genomes of Rhodothermaceae. Strain RA has genes for sulfate permease and arylsulfatase to withstand the high sulfur and sulfate contents of the hot spring. Putative genes encoding proteins involved in adaptation to osmotic stress were identified which encode proteins namely Na+/H+ antiporters, a sodium/solute symporter, a sodium/glutamate symporter, trehalose synthase, malto-oligosyltrehalose synthase, choline-sulfatase, potassium uptake proteins (TrkA and TrkH), osmotically inducible protein C, and the K+ channel histidine kinase KdpD. Furthermore, genome description of strain RA and comparative genome studies in relation to other related genera provide an overview of the uniqueness of this bacterium. PMID:27471502

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ28. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved

Five strains of proteolytic rumen bacteria were treated with condensed tannins (CT) purified from Lotus pedunculatus and Lotus corniculatus to investigate their effect on the growth of these bacteria in vitro. Streptococcus bovis NCFB 2476, Eubacterium sp. C124b, Prevotella bryantii B14, Butyrivibrio fibrisolvens H17c, and Clostridium proteoclasticum B316T were tested against 200, 400, and 600 microg CT x mL(-1) extracted from L. pedunculatus and L. corniculatus. In the absence of CT, all bacterial strains showed typical growth and reached maximum optical density (OD) after 6-8 h of incubation in a plant protein medium. Growth of Eubacterium sp., P. bryantii, and B. fibrisolvens was inhibited (P < 0.01-0.001) more by the CT from L. pedunculatus than by the CT from L. corniculatus. All strains continued to grow in the presence of 200 microg x mL(-1) of the CT from L. pedunculatus, but attained significantly (P < 0.05-0.01) lower maximum OD600 values than (minus CT) controls, except for S. bovis. At 400 and 600 microg x mL(-1), the addition of CT from L. pedunculatus inhibited (P < 0.05-0.001) the growth of all bacterial strains tested compared with controls. The growth of Eubacterium sp. and P. bryantii was stimulated for the first 4-6 h of incubation (P < 0.001) by 200 microg x mL(-1) of CT from L. corniculatus, but then declined leading to a significant difference in OD values compared with the controls. At 400 microg x mL(-1), the CT from L. corniculatus reduced (P < 0.05-0.01) the growth of all strains except S. bovis, while 600 microg x mL(-1) inhibited (P < 0.01-0.001) the growth of all strains. To study the mechanism of CT action, the degradation of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; Fraction 1 Leaf protein) was followed after bacterial cells or Rubisco were preincubated with CT extracted from L. corniculatus and L. pedunculatus. Both preincubations decreased LSU degradation, but they differed in their

Little is known regarding the range of conditions in which many Perkinsus species may proliferate, making it difficult to predict conditions favorable for their expansion, to identify conditions inducing mortality, or to identify instances of potential cross-infectivity among sympatric host species. In this study, the effects of salinity on viability, metabolic activity and proliferation of P. marinus, P. olseni and P. chesapeaki were determined. Specifically, this research examined the effects of 5 salinities (7, 11, 15, 25, 35???), (1) without acclimation, on the viability and metabolic activity of 2 isolates of each Perkinsus species, and (2) with acclimation, on the viability, metabolic activity, size and number of 1 isolate of each species. P. chesapeaki showed the widest range of salinity tolerance of the 3 species, with high viability and cell proliferation at all salinities tested. Although P. chesapeaki originated from low salinity areas (i.e. <15???), several measures (i.e. cell number and metabolic activity) indicated that higher salinities (15, 25???) were more favorable for its growth. P. olseni, originating from high salinity areas, had better viability and proliferation at the higher salinities (15, 25, 35???). Distinct differences in acute salinity response of the 2 P. olseni isolates at lower salinities (7, 11???), however, suggest the need for a more expansive comparison of isolates to better define the lower salinity tolerance. Lastly, P. marinus was more tolerant of the lower salinities (7 and 11???) than P. olseni, but exhibited reduced viability at 7???, even after acclimation. ?? Inter-Research 2006.

Cyanobacteria, including members of the genus Prochlorococcus, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. However, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is not known. Complicating the study of cyanobacterial carboxysomes has been the inability to obtain homogeneous carboxysome preparations. This study describes the first successful purification and characterization of carboxysomes from the marine cyanobacterium Prochlorococcus marinus MED4. Because the isolated P. marinus MED4 carboxysomes were free from contaminating membrane proteins, their protein complement could be assessed. In addition to the expected shell proteins, the CsoS1D protein that is not encoded by the canonical cso gene clusters of α-cyanobacteria was found to be a low-abundance shell component. This finding and supporting comparative genomic evidence have important implications for carboxysome composition, structure, and function. Our study indicates that carboxysome composition is probably more complex than was previously assumed based on the gene complements of the classical cso gene clusters.

Patterns of genetic variation for the tick Amblyomma dissimile were analyzed from a total of 200 ticks collected on 12 toads (Bufo marinus), 14 snakes (Boa constrictor), and 8 lizards (Iguana iguana) at 11 localities. The analyses were performed on electrophoretic data from 8 isozyme loci. Mean heterozygosity per locus was 6% (+/-3.1) per population. Differences in allelic frequencies among ticks from different individual hosts were the major source of genetic variability in this study. Host species was a smaller source of genetic variation. Genetic distances between localities varied according to which host species was present in each locality, and these appeared to be related to the extent of habitat overlap between host species. The smallest genetic distances between samples from different host species were recorded for I. iguana and B. constrictor. In contrast, the genetic distances between tick samples from B. marinus and either of the reptile species were significantly larger than between tick samples from this amphibian species. Ecological variables or the geographic distance did not explain the local patterns of differentiation observed in A. dissimile. Major genetic differences between island and mainland sites (0.03702) suggested an association between genetic distances and geographic isolation. The consistency between patterns of genetic variation and those of host home range overlap suggests that host dispersion is the main force structuring the genetic variation within this tick species.

Major degraders of petroleum hydrocarbons in tropical seas have been indicated only by laboratory culturing and never through observing the bacterial community structure in actual environments. To demonstrate the major degraders of petroleum hydrocarbons spilt in actual tropical seas, indigenous bacterial community in seawater at Sentosa (close to a port) and East Coast Park (far from a port) in Singapore was analyzed. Bacterial species was more diverse at Sentosa than at the Park, and the composition was different: γ-Proteobacteria (57.3%) dominated at Sentosa, while they did not at the Park. Specialized hydrocarbonoclastic bacteria (SHCB), which use limited carbon sources with a preference for petroleum hydrocarbons, were found as abundant species at Sentosa, indicating petroleum contamination. On the other hand, SHCB were not the abundant species at the Park. The abundant species of SHCB at Sentosa were Oleibacter marinus and Alcanivorax species (strain 2A75 type), which have previously been indicated by laboratory culturing as important petroleum-aliphatic-hydrocarbon degraders in tropical seas. Together with the fact that SHCB have been identified as major degraders of petroleum hydrocarbons in marine environments, these results demonstrate that the O. marinus and Alcanivorax species (strain 2A75 type) would be major degraders of petroleum aliphatic hydrocarbons spilt in actual tropical seas. PMID:23824553

The oyster Crassostrea gasar is a species widely used as food and a source of income for the local population of the estuaries of Northeast Brazil. Perkinsus marinus and Perkinsus olseni are deleterious parasites for oyster farming and were recently detected in Brazil. In this study, a histopathologic survey of the oyster C. gasar cultured in the estuary of the River Mamanguape (Paraíba State) was performed. Adult oysters were collected in December 2011 and March, May, August and October 2012 and processed for histology and Perkinsus sp. identification by molecular analyses. Histopathological analysis revealed the presence of parasitic organisms including viral gametocytic hypertrophy, prokaryote-like colonies, protozoans (Perkinsus sp. and Nematopsis sp.) and metazoans (Tylocephalum sp. and cestodes). Other commensal organisms were also detected (the protozoan Ancistrocoma sp. and the turbellarian Urastoma sp.). The protozoan parasite Perkinsus sp. had the highest overall prevalence among the symbiotic organisms studied (48.9%), followed by Nematopsis sp. (36.3%). The other organisms were only sporadically observed. Only the protozoan Perkinsus sp. caused alterations in the oysters' infected organs. Molecular analyses confirmed the presence of P. marinus, P. olseni and Perkinsus beihaiensis infecting the oyster C. gasar. This is the first report of P. beihaiensis in this oyster species.

Prochlorococcus marinus CCMP 1375, a ubiquitous and ecologically important marine prochlorophyte, was bound to possess functional genes coding for the alpha and beta subunits of a phycobiliprotein. The latter is similar to phycoerythrins (PE) from marine Synechococcus cyanobacteria and bind a phycourobilin-like pigment as the major chromophore. However, differences in the sequences of the alpha and beta chains compared with known PE subunits and the presence of a single bilin attachment site on the alpha subunit designate it as a novel PE type, which we propose naming PE-III. P. marinus is the sole prokaryotic organisms known so far that contains chlorophylls a and b as well as phycobilins. These data strongly suggest that the common ancestor of prochlorophytes and the Synechococcus cyanobacteria contained phycobilins. Flow cytometric data from the tropical Pacific Ocean provide evidence that deep populations of Prochlorococcus possess low amounts of a PE-like pigment, which could serve either in light harvesting or nitrogen storage or both. Images Fig. 2 Fig. 4 PMID:8855320

Since 1937, thirteen species of non-indigenous anurans have made their way to Guam. Of these, at least six have established breeding populations. Various pathways led to the introduction of these species to the island. The only anuran intentionally introduced was Chaunus marinus (formerly Bufo marinus), which was brought to Guam as a biocontrol agent. Kaloula picta, K. pulchra, Polypedates leucomystax, and probably Litoria fallax arrived as stowaways via maritime or air-transport vessels. Eleutherodactylus coqui and Euhyas (formerly Eleutherodactylus) planirostris appear to have entered Guam through the horticultural trade. Specimens of Pseudacris regilla were found among agricultural products and Christmas trees. Five species have been transported to Guam via the aquacultural trade. The importation of tilapia, milkfish, and white shrimp from China, Hong Kong, Taiwan, and the Philippines was associated with the introduction to Guam of Fejervarya cancrivora, F. limnocharis sensu lato, Microhyla pulchra, Polypedates megacephalus, and Sylvirana guentheri (formerly Rana guentheri). Presently, no quarantine or containment guidelines have been established for Guam's aquacultural industry. ?? 2007 The Authors.

The pleasure oyster Crassostrea corteziensis is collected and cultured in Nayarit on the Pacific coast of Mexico, and the improvement and promotion of its culture are seen as a possible source for the economic development of coastal populations. However, information about the parasite fauna of the pleasure oyster is almost completely lacking. A histopathological survey carried out in two estuaries, Boca del Camichín and Pozo Chino, revealed the presence of hypertrophied gametes, rickettsiales-like prokaryotes (RLPs), the protozoan Perkinsus marinus, a protozoan Nematopsis sp., Ancistrocoma-like ciliates (ALCs), Sphenophrya-like ciliates, a turbellarian Urastoma sp., and encysted crustaceans. In general, prevalence and intensity of parasites were similar in both localities except that ALCs and encysted crustaceans were more prevalent in Pozo Chino than in Boca del Camichín. Perkinsus marinus and RLPs seem to represent a more significant risk for the health of pleasure oysters than do the other parasites, and surveillance and control of these parasites are needed for the development of pleasure oyster culture.

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M.

Tetrachloroethene is a prominent groundwater pollutant that can be reductively dechlorinated by mixed anaerobic microbial populations to the nontoxic product ethene. Strain 195, a coccoid bacterium that dechlorinates tetrachlorethene to ethene, was isolated and characterized. Growth of strain 195 with H{sub 2} and tetrachloroethene as the electron donor and acceptor pair required extracts from mixed microbial cultures. Growth of strain 195 was resistant to ampicillin and vancomycin; its cell wall did not react with a peptidoglycan-specific lectin and its ultrastructure resembled S-layers of Archaea. Analysis of the 16S ribosomal DNA sequence of strain 195 indicated that it is a eubacterium without close affiliation to any known groups. 24 refs., 4 figs., 1 tab.

Mycoplasma pneumoniae is a pathogenic eubacterium lacking a cell wall. Three decades ago, a "rod", an intracellular cytoskeletal structure, was discovered that was assumed to define and stabilize the elongated cell shape. Later, by treatment with detergent, a "Triton shell" (i.e. a fraction of detergent-insoluble cell material) could be obtained, believed to contain additional cytoskeletal elements. Now, by application of a modified Triton X-100 treatment, we are able to demonstrate that M. pneumoniae possesses a cytoskeleton consisting of a blade-like rod and a peripheral lining located close to the inner face of the cytoplasmic membrane, exhibiting features of a highly regular network. Attached "stalks" may support the cytoplasmic membrane. The rod was connected to the cell periphery by "spokes" and showed a defined ultrastructure. Its proximal end was found to be attached to a wheel-like complex. Fibrils extended from the proximal end of the rod into the cytoplasm.

Pyridoxal-5′-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases. PMID:24888348

Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.

In this paper, the chemical composition and the antimicrobial activity of the essential oil of Angelica archangelica L. (Apiaceae) roots from central Italy were analyzed. The major constituents of the oil were α-pinene (21.3%), δ-3-carene (16.5%), limonene (16.4%) and α-phellandrene (8.7%). The oil shows a good antimicrobial activity against Clostridium difficile, Clostridium perfringens, Enterococcus faecalis, Eubacterium limosum, Peptostreptococcus anaerobius, and Candida albicans with minimum inhibitory concentration (MIC) values of 0.25, 0.25, 0.13, 0.25, 2.25, and 0.50% v/v, respectively. A weaker antimicrobial activity against bifidobacteria and lactobacilli-very useful in the intestinal microflora-has also been shown with MIC values >4.0% v/v.

The complete genome of the green-sulfur eubacterium Chlorobium tepidum TLS was determined to be a single circular chromosome of 2,154,946 bp. This represents the first genome sequence from the phylum Chlorobia, whose members perform anoxygenic photosynthesis by the reductive tricarboxylic acid cycle. Genome comparisons have identified genes in C. tepidum that are highly conserved among photosynthetic species. Many of these have no assigned function and may play novel roles in photosynthesis or photobiology. Phylogenomic analysis reveals likely duplications of genes involved in biosynthetic pathways for photosynthesis and the metabolism of sulfur and nitrogen as well as strong similarities between metabolic processes in C. tepidum and many Archaeal species. PMID:12093901

The in vitro inhibitory activity of tinidazole, metronidazole, ornidazole, cefoxitin and moxalactam was determined against 150 isolates of clinically important anaerobes including Bacteroides fragilis, Bacteroides bivius and Clostridium perfringens by means of agar dilution tests. The members of 18 gramnegative and 14 grampositive species were inhibited by tinidazole at less than or equal to 0,01-8 micrograms/ml thus being without exception susceptible to the drug. A similar in vitro activity was recorded for metronidazole and ornidazole. Though cefoxitin and moxalactam generally had a good in vitro activity against most anaerobic species, single strains of Bacteroides thetaiotaomicron, B. distasonis, Clostridium difficile and Eubacterium rectale were resistant to these drugs. With regard to its in vitro activity, tinidazole seems to be a promising substance for the therapy of anaerobic infections.

A microbiological survey has been carried out on 179 healthy, child-bearing aged, non-pregnant women, with the aim to evaluate the incidence of anaerobic non-spore forming bacteria in the normal vaginal flora. This group of bacteria has been isolated in 50.3% of women, with a clear prevalence of "anaerobic Streptococci " and Bacteroides, followed by Fusobacterium and Veillonella. No Propionibacterium, Eubacterium or Bifidobacterium have been isolated. According to many Authors the non-spore forming anaerobes must be considered opportunistic bacteria, responsible of many infections of the female genital tract, especially when associated with other aerobic or facultative bacteria. Antibiograms have demonstrated a wide spectrum of activity of chloramphenicol and clindamycin; although not widely distributed, antibacterial activity have also shown metronidazole, penicillins, cephalosporins and lincomycin.

A live, in-situ metabolomics capability was developed for prokaryotic cultures under controlled growth conditions. Toward this goal, a radiofrequency-transparent bioreactor was developed and integrated with a commercial wide-bore nuclear magnetic resonance (NMR) imaging spectrometer and a commercial bioreactor controller. Water suppressed 1H NMR spectroscopy was used to monitor glucose and fructose utilization and byproduct excretion by Eubacterium aggregans (an anaerobic bacterial species relevant for biofuel production) under controlled batch and continuous culture conditions. The resulting metabolite profiles (short chain organic acids and ethanol) and trends are consistent with existing knowledge of its metabolism. However, our study also showed that E. aggregans produces lactate end product in significant concentrations—a result not previously reported. The advantages of live in-situ microbial metabolomics analysis and its complementariness with functional genomics/systems biology methods are discussed.

Ionizing Radiation (IR) Resistance in Bacteria. Until recently, there have been no clear physiologic predictors of a cell's ability to recover from ionizing radiation (IR) and other DOE-relevant oxidative stress conditions. In general, the most resistant bacteria have been Gram-positive (e.g., Deinococcus, Arthrobacter, Lactobacillus & Enterococcus spp.) and the most sensitive have been Gram-negative (e.g., Pseudomonas, Shewanella & Neisseria spp.). However, there are several reported exceptions to this paradigm, the Gram-negative cyanobacterium Chroococcidiopsis is extremely resistant to IR, whereas the Gram-positive Micrococcus luteus is sensitive. We have identified biomolecular signatures for radiation sensitivity and resistance which are independent of phylogeny, where very high and very low intracellular Mn/Fe concentration ratios correlated with very high and very low resistances, respectively; and restricting Mn(II) in the famously resistant Deinococcus radiodurans sensitized this eubacterium to IR.

Until recently, there have been no clear physiologic predictors of a cell's ability to recover from ionizing radiation (IR), desiccation, and other DOE-relevant oxidative stress conditions. In general, the most resistant bacteria have been Gram-positive (e.g., Deinococcus, Arthrobacter, Lactobacillus & Enterococcus spp.) and the most sensitive have been Gram-negative (e.g., Pseudomonas, Shewanella & Neisseria spp.). However, there are several reported exceptions to this paradigm, the Gram-negative cyanobacterium Chroococcidiopsis is extremely resistant to IR, whereas the Gram-positive Micrococcus luteus is sensitive. We have identified biomolecular signatures for radiation sensitivity and resistance which are independent of phylogeny, where very high and very low intracellular Mn/Fe concentration ratios correlated with very high and very low resistances, respectively; and restricting Mn(II) in the famously resistant Deinococcus radiodurans sensitized this eubacterium to IR (http://cfyn.ifas.ufl.edu/radiation.pdf).

Thermotoga maritima is one of the more unusual eubacteria: It is highly thermophilic, growing at temperatures higher than any other eubacterium; its cell wall appears to have a unique structure and its lipids a unique composition; and the organism is surrounded by a loose-fitting sheath of unknown function. Its phenotypic uniqueness is matched by its phylogenetic position; Thermotoga maritima represents the deepest known branching in the eubacterial line of descent, as measured by ribosomal RNA sequence comparisons. T. maritima also represents the most slowly evolving of eubacterial lineages. The fact that the two deepest branchings in the eubacterial line of descent (the other, the green non-sulfur bacteria and relatives, i.e. Chloroflexus, Thermomicrobium, etc.) are both basically thermophilic and slowly evolving, strongly suggests that all eubacteria have ultimately arisen from a thermophilic ancestor.

The in vitro fermentation selectivity of hydrolyzed caseinomacropeptide (CMP) glycosylated, via Maillard reaction (MR), with lactulose, galacto-oligosaccharides from lactose (GOSLa), and galacto-oligosaccharides from lactulose (GOSLu) was evaluated, using pH-controlled small-scale batch cultures at 37 °C under anaerobic conditions with human feces. After 10 and 24 h of fermentation, neoglyconjugates exerted a bifidogenic activity, similar to those of the corresponding prebiotic carbohydrates. No significant differences were found in Bacteroides , Lactobacillus - Enterococcus , Clostridium histolyticum subgroup, Atopobium and Clostridium coccoides - Eubacterium rectale populations. Concentrations of lactic acid and short-chain fatty acids (SCFA) produced during the fermentation of prebiotic carbohydrates were similar to those produced for their respective neoglycoconjugates at both fermentation times. These findings, joined with the functional properties attributed to CMP, could open up new applications of MR products involving prebiotics as novel multiple-functional ingredients with potential beneficial effects on human health.

The agar dilution method was used to determine the inhibitory activities of 28 antimicrobial agents against 35 strains of the genus Peptostreptococcus, 4 strains of the species Peptococcus niger, 20 strains of the species Megasphaera elsdenii, 7 strains from the species Acidaminococcus fermentans, 8 strains of the genus Clostridium, 11 strains of the genus Eubacterium, and 1 strain of the species Propionibacterium acidipropionici, all of which were isolated from 125 clinical cases of ovine foot rot between January 1987 and December 1988. The three unreidopenicillins studied proved to be the most active antimicrobial agents, with a high percentage of strains being susceptible at a concentration of 64 micrograms/ml. Penicillin G, ampicillin, and the three cephalosporins studied also had good activity. Fosfomycin showed a high degree of activity among the 116 anaerobic bacteria tested. PMID:1590689

A dot-blot hybridisation assay using high molecular weight DNA as whole chromosomal probes was used to differentiate Bacteroides tectum from Bacteroides fragilis. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes. The whole chromosomal probes were specific--differentiating B. tectum from B. fragilis and both from a variety of other species (including other members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. However, even at very high stringencies, B. tectum homology groups I, II and III were not distinguishable from one another using either 32P-labelled or DIG-labelled probes. Thus, DIG-labelled whole chromosome probes directed against cellular DNA released directly onto nitrocellulose membranes is considered a useful method for diagnostic veterinary laboratories wishing to identify B. tectum and distinguish it from B. fragilis and other oral anaerobic flora of cats.

A corrinoid screening of several phylogenetically diverse ``archaebacteria'' revealed vitamin B12-like corrinoids. This indicates an optimized structure and function relationship of the corrinoids under different bacterial growth conditions during the early evolution of live. Some of these corrinoids have been substituted by modified corrinoids in growing cells without affecting the generation times of the bacteria. In this respect, the discovery of the unique para-cresolyl cobamide from the eubacterium Sporomusa ovata attracted attention. The unusual structure of this corrinoid was achieved by a biosynthesis proceeding via a novel and stereospecific activation mechanism of aromatic compounds. The corrinoid was detected both in the membrane fraction and in the soluble fraction of the cells. Methyltransfer is one of the probable functions of the para-cresolyl cobamide in Sporomusa.

The gut bacteria exert phenotypic traits to the host but the factors which determine the gut bacterial profile (GBP) is poorly understood. This study aimed to understand the effect of ethnicity and geography on GBP of Mongoloid and Proto-Australoid tribes of India. Fecal bacterial diversity was studied in fifteen tribal populations representing four geographic regions (Assam, Telangana, Manipur and Sikkim) by DGGE followed by NGS analysis on Illumina MiSeq platform. Geography and diet had significant effect on GBP of the Indian tribes which was dominated by Prevotella. The effects were more prominent with lower taxonomic levels, indicating probable functional redundancy of the core GBP. A comparison with the worldwide data revealed that GBP of the Indian population was similar to the Mongolian population (Mongolia). The bacterial genera Faecalibacterium, Eubacterium, Clostridium, Blautia, Ruminococcus and Roseburia were found to be core genera in the representative populations of the world. PMID:26689136

The presence of rhodanese activity has been investigated in two strains of thermophilic eubacteria and two strains of extremophiles. Bacillus acidocaldarius, a thermoacidophilic eubacterium, showed the highest levels of enzyme activity. Whole cells, previously subjected to one cycle of freeze-thawing, were immobilized by entrapment in the polysaccharide matrices Ca-alginate, {kappa}-carrageenan and chitosan, and in an insolubilized gelatin gel. The results obtained with the different immobilizates in terms of activity yield, possibility of regeneration and operative stability were evaluated with the aim of setting up a continuous system. This was achieved with a system consisting of B. acidocaldarius cells entrapped in an insolubilized gelatin matrix. The latter, in the form of a thin membrane, was employed in a custom-conceived reactor operating as a plug flow reactor. 21 refs., 3 figs., 2 tabs.

Tonsilloliths are a potential cause of oral malodor. In this study, microbial profiles and composition of tonsilloliths were determined using culture-independent molecular methods and scanning electron microscopy. 16S ribosomal RNA bacterial genes (16S rDNAs) isolated from tonsilloliths of 6 individuals were amplified by PCR and cloned into Escherichia coli. Partial 16S rDNA sequences of approximately 600 bases of cloned inserts were used to determine species identity by comparison with sequences of known species. Characteristics of bacteria on the surface and inside the tonsillolith were analyzed using scanning electron microscopy. Anaerobic bacteria detected in tonsilloliths belonged to the genera Eubacterium, Fusobacterium, Megasphaera, Porphyromonas, Prevotella, Selenomonas and Tannerella, all of which appear to be associated with production of volatile sulfur compounds. Electron microscopy revealed cocci and rods on the surface and rods predominating inside the tonsilloliths. These results support the tonsillolith as an origin of oral malodor.

The mechanism by which acidophilic bacteria generate and maintain their cytoplasmic pH close to neutrality was investigated. For this purpose we determined the components of proton motive force in the eubacterium Bacillus acidocaldarius and the archaebacterium Thermoplasma acidophilum. After correction for probe binding, the proton motive force of untreated cells was 190 to 240 mV between external pH 2 and 4. Anoxia diminished total proton motive force and the transmembrane pH difference by 60 to 80 mV. The protonophore 2,4-dinitrophenol abolished the total proton motive force almost completely and diminished the transmembrane pH difference by at least two units. However, even after correction for probe binding, protonophore-treated cells maintained a pH difference of approximately one unit. PMID:2981803

Three enriched cultures (48, 15a, and 26a), selected from more than 80 soil and water samples, could grow anaerobically in the presence of crude oil at 30{degrees}C and could ferment molasses to gases and organic acids. Oil recovery by culture 48 in the laboratory model experiment was enhanced by 25.2% over the original reserves and by 53.7% over the residual reserves. Enriched culture 48 was composed of at least 4 species belonging to the genera Eubacterium, Fusobacterium, and Bacteroides. This enriched culture was used as inoculum for MEOR field trials at Jilin oil field with satisfactory results. The importance of the role of these isolates in EOR was confirmed by their presence and behavior in the fluids produced from the microbiologically treated reservoir.

Group II introns are self-splicing ribozymes that are essential in many organisms, and they are hypothesized to share a common evolutionary ancestor with the spliceosome. While structural similarity of RNA components supports this connection, it is of interest to determine whether associated protein factors also share an evolutionary heritage. Here we present the crystal structures of reverse transcriptase (RT) domains from two group II intron encoded proteins (maturases) from Roseburia intestinalis and Eubacterium rectale, obtained at 1.2 Å and 2.1 Å respectively. Their architecture is more similar to the spliceosomal Prp8 RT-like domain than to any other RTs, and they share substantial similarity with flaviviral RNA polymerases. The RT domain itself is sufficient for binding intron RNA with high affinity and specificity, and it is contained within an active RT enzyme. These studies provide a foundation for understanding structure-function relationships within group II intron–maturase complexes. PMID:27136328

Introduction In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. Methods Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. Results Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced. Conclusions This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a ‘systemic disease’, linking the lung and the gut in a joined axis. PMID:23613805

Pyrosequencing analysis of intestinal microflora from healthy Thai vegetarians and non-vegetarians exhibited 893 OTUs covering 189 species. The strong species indicators of vegetarians and non-vegetarians were Prevotella copri and Bacteroides vulgatus as well as bacteria close to Escherichia hermanii with % relative abundance of 16.9 and 4.5-4.7, respectively. Core gut microbiota of the vegetarian and non-vegetarian groups consisted of 11 and 20 different bacterial species, respectively, belonging to Actinobacteria, Firmicutes, and Proteobacteria commonly found in both groups. Two species, Faecalibacterium prausnitzii and Gemmiger formicilis, had a prevalence of 100% in both groups. Three species, Clostridium nexile, Eubacterium eligens, and P. copri, showed up in most vegetarians, whereas more diversity of Collinsella aerofaciens, Ruminococcus torques, various species of Bacteroides, Parabacteroides, Escherichia, and different species of Clostridium and Eubacterium were found in most non-vegetarians. Considering the correlation of personal characters, consumption behavior, and microbial groups, the age of non-vegetarians showed a strong positive correlation coefficient of 0.54 (p = 0.001) to Bacteroides uniformis but exhibited a moderate one to Alistipes finegoldii and B. vulgatus. Only a positive moderate correlation of body mass index and Parabacteroides distasonis appeared. Based on the significant abundance of potential pathogens, the microbiota of the non-vegetarian group showed an abundance of potential pathogen varieties of Bilophila wadsworthia, Escherichia coli, and E. hermannii, whereas that of the vegetarian group served for only Klebsiella pneumoniae. These results implied that the microbiota of vegetarians with high abundance of P. copri and low potential pathogen variety would be a way to maintain good health in Thais.

The microbial community of the human colon contains many bacteria that produce lactic acid, but lactate is normally detected only at low concentrations (<5 mM) in feces from healthy individuals. It is not clear, however, which bacteria are mainly responsible for lactate utilization in the human colon. Here, bacteria able to utilize lactate and produce butyrate were identified among isolates obtained from 10(-8) dilutions of fecal samples from five different subjects. Out of nine such strains identified, four were found to be related to Eubacterium hallii and two to Anaerostipes caccae, while the remaining three represent a new species within clostridial cluster XIVa based on their 16S rRNA sequences. Significant ability to utilize lactate was not detected in the butyrate-producing species Roseburia intestinalis, Eubacterium rectale, or Faecalibacterium prausnitzii. Whereas E. hallii and A. caccae strains used both D- and L-lactate, the remaining strains used only the d form. Addition of glucose to batch cultures prevented lactate utilization until the glucose became exhausted. However, when two E. hallii strains and one A. caccae strain were grown in separate cocultures with a starch-utilizing Bifidobacterium adolescentis isolate, with starch as the carbohydrate energy source, the L-lactate produced by B. adolescentis became undetectable and butyrate was formed. Such cross-feeding may help to explain the reported butyrogenic effect of certain dietary substrates, including resistant starch. The abundance of E. hallii in particular in the colonic ecosystem suggests that these bacteria play important roles in preventing lactate accumulation.

A total of 650 ticks, including 13 species from five genera, were collected from animals, from people, or by flagging of the vegetation at sites on the Thai-Myanmar border and in Vietnam. They were tested by PCR to detect DNA of bacteria of the order RICKETTSIALES: Three Anaplasma spp. were detected in ticks collected in Thailand, including (i) Anaplasma sp. strain AnDa465, which was considered a genotype of Anaplasma platys (formerly Ehrlichia platys) and which was obtained from Dermacentor auratus ticks collected from dogs; (ii) Anaplasma sp. strain AnAj360, which was obtained from Amblyomma javanense ticks collected on a pangolin; and (iii) Anaplasma sp. strain AnHl446, which was closely related to Anaplasma bovis and which was detected in Haemaphysalis lagrangei ticks collected from a bear. Three Ehrlichia spp. were identified, including (i) Ehrlichia sp. strain EBm52, which was obtained from Boophilus microplus ticks collected from cattle from Thailand; (ii) Ehrlichia sp. strain EHh324, which was closely related to Ehrlichia chaffeensis and which was detected in Haemaphysalis hystricis ticks collected from wild pigs in Vietnam; and (iii) Ehrlichia sp. strain EHh317, which was closely related to Ehrlichia sp. strain EBm52 and which was also detected in H. hystricis ticks collected from wild pigs in Vietnam. Two Rickettsia spp. were detected in Thailand, including (i) Rickettsia sp. strain RDla420, which was detected in Dermacentor auratus ticks collected from a bear, and (ii) Rickettsia sp. strain RDla440, which was identified from two pools of Dermacentor larvae collected from a wild pig nest. Finally, two bacteria named Eubacterium sp. strain Hw124 and Eubacterium sp. strain Hw191 were identified in Haemaphysalis wellingtoni ticks collected from chicken in Thailand; these strains could belong to a new group of bacteria.

Thermoanaerobacter tengcongensis is a rod-shaped, gram-negative, anaerobic eubacterium that was isolated from a freshwater hot spring in Tengchong, China. Using a whole-genome-shotgun method, we sequenced its 2,689,445-bp genome from an isolate, MB4(T) (Genbank accession no. AE008691). The genome encodes 2588 predicted coding sequences (CDS). Among them, 1764 (68.2%) are classified according to homology to other documented proteins, and the rest, 824 CDS (31.8%), are functionally unknown. One of the interesting features of the T. tengcongensis genome is that 86.7% of its genes are encoded on the leading strand of DNA replication. Based on protein sequence similarity, the T. tengcongensis genome is most similar to that of Bacillus halodurans, a mesophilic eubacterium, among all fully sequenced prokaryotic genomes up to date. Computational analysis on genes involved in basic metabolic pathways supports the experimental discovery that T. tengcongensis metabolizes sugars as principal energy and carbon source and utilizes thiosulfate and element sulfur, but not sulfate, as electron acceptors. T. tengcongensis, as a gram-negative rod by empirical definitions (such as staining), shares many genes that are characteristics of gram-positive bacteria whereas it is missing molecular components unique to gram-negative bacteria. A strong correlation between the G + C content of tDNA and rDNA genes and the optimal growth temperature is found among the sequenced thermophiles. It is concluded that thermophiles are a biologically and phylogenetically divergent group of prokaryotes that have converged to sustain extreme environmental conditions over evolutionary timescale.

Abstract Accurate stock assessments for each of the dominant species of sand lances in the northeast Atlantic Ocean and adjacent areas are not available due to the lack of a reliable identification procedure; therefore, appropriate measures of fisheries management or conservation of sand lances cannot be implemented. In this study, detailed morphological and molecular features are assessed to discriminate between four species of sand lances belonging to the genera Ammodytes and Hyperoplus. Morphological characters described by earlier authors as useful for identification of the genera are confirmed, and two additional distinguishing characters are added. A combination of the following morphological characters is recommended to distinguish between the genera Hyperoplus and Ammodytes: the protrusibility of the premaxillae, the presence of hooked ends of the prevomer, the number of dermal plicae, and the pectoral-fin length as a percentage of the standard length. The discriminant function analysis revealed that morphometric data are not very useful to distinguish the species of each of the two genera. The following meristic characters improve the separation of Hyperoplus lanceolatus from Hyperoplus immaculatus: the number of lower arch gill rakers, total number of gill rakers, numbers of caudal vertebrae and total vertebrae, and numbers of dorsal-fin and anal-fin rays. It is confirmed that Ammodytes tobianus differs from Ammodytes marinus by its belly scales that are organised in tight chevrons, scales which are present over the musculature at the base of the caudal fin, as well as by the lower numbers of dermal plicae, dorsal-fin rays, and total vertebrae. In contrast to the morphological data, mitochondrial COI sequences (DNA barcodes) failed to separate unambiguously the four investigated species. Ammodytes tobianus and Hyperoplus lanceolatus showed an overlap between intraspecific and interspecific K2P genetic distances and cannot be reliably distinguished using

Accurate stock assessments for each of the dominant species of sand lances in the northeast Atlantic Ocean and adjacent areas are not available due to the lack of a reliable identification procedure; therefore, appropriate measures of fisheries management or conservation of sand lances cannot be implemented. In this study, detailed morphological and molecular features are assessed to discriminate between four species of sand lances belonging to the genera Ammodytes and Hyperoplus. Morphological characters described by earlier authors as useful for identification of the genera are confirmed, and two additional distinguishing characters are added. A combination of the following morphological characters is recommended to distinguish between the genera Hyperoplus and Ammodytes: the protrusibility of the premaxillae, the presence of hooked ends of the prevomer, the number of dermal plicae, and the pectoral-fin length as a percentage of the standard length. The discriminant function analysis revealed that morphometric data are not very useful to distinguish the species of each of the two genera. The following meristic characters improve the separation of Hyperoplus lanceolatus from Hyperoplus immaculatus: the number of lower arch gill rakers, total number of gill rakers, numbers of caudal vertebrae and total vertebrae, and numbers of dorsal-fin and anal-fin rays. It is confirmed that Ammodytes tobianus differs from Ammodytes marinus by its belly scales that are organised in tight chevrons, scales which are present over the musculature at the base of the caudal fin, as well as by the lower numbers of dermal plicae, dorsal-fin rays, and total vertebrae. In contrast to the morphological data, mitochondrial COI sequences (DNA barcodes) failed to separate unambiguously the four investigated species. Ammodytes tobianus and Hyperoplus lanceolatus showed an overlap between intraspecific and interspecific K2P genetic distances and cannot be reliably distinguished using the common

There are two adult life history types among lamprey species, nonparasitic and parasitic, with the former commencing the final interval of sexual maturation immediately after metamorphosis. There are no extensive studies that directly compare hormone profiles during the life cycles of nonparasitic and parasitic lamprey species, yet such data may explain differences in development, reproductive maturation, and feeding status. The present study uses immunohistochemistry to show the life cycle profiles for gonadotropin-releasing hormones (GnRH-I and -III) in the brain of the nonparasitic species, the American brook lamprey, Lampetra appendix, for comparison with the extensive, published, immunohistochemical data on these hormones in the parasitic species, the sea lamprey, Petromyzon marinus. The complete cDNAs for the two lamprey prohormones, proopiocortin (POC), and proopiomelanotropin (POM), were cloned for L. appendix and both nucleotide and deduced amino acid sequences were compared with those previously published for P. marinus. The POC and POM cDNAs for both species were used in expression studies, with Northern blotting, throughout their life cycles. Although GnRH-I and -III immunohistochemistry revealed a similar distribution of immunoreactive cells and fibers in the two species during the life cycles, a qualitative evaluation of staining intensity in L. appendix, implied early activity in the brains of metamorphosis of this species, particularly in GnRH-I. GnRH-III seems to be important in larval life and early metamorphosis in both species. A novel feature of this immunohistochemical study is the monthly observations of the distribution and relative intensity of the two GnRHs during the critical period of final sexual maturation that lead to spawning and then the spent animal. L. appendix POC and POM nucleotide sequences had 92.9 and 94.6% identity, respectively, with P. marinus POC and POM and there was an earlier increase in their expression during

A 15 h ozonation was performed on bioremediated soil to remove recalcitrant residual oil. To monitor the survival of indigenous microorganisms in the soil during in-situ chemical oxidation(ISCO) culturing and a functional genearray, GeoChip, was used to examine the functional genes and structure of the microbial community during ozonation (0h, 2h, 4h, 6h, 10hand15h). Breakthrough ozonation decreased the population of cultivable heterotrophic bacteria by about 3 orders of magnitude. The total functional gene abundance and diversity decreased during ozonation, as the number of functional genes was reduced by 48percent after 15 h. However, functional genes were evenly distributed during ozonation as judged by the Shannon-Weaver Evenness index. A sharp decrease in gene number was observed in the first 6 h of ozonation followed by a slower decrease in the next 9 h, which was consistent with microbial populations measured by a culture based method. Functional genes involved in carbon, nitrogen, phosphors and sulfur cycling, metal resistance and organic remediation were detected in all samples. Though the pattern of gene categories detected was similar for all time points, hierarchica lcluster of all functional genes and major functional categories all showed a time-serial pattern. Bacteria, archaea and fungi decreased by 96.1percent, 95.1percent and 91.3percent, respectively, after 15 h ozonation. Delta proteobacteria, which were reduced by 94.3percent, showed the highest resistance to ozonation while Actinobacteria, reduced by 96.3percent, showed the lowest resistance. Microorganisms similar to Rhodothermus, Obesumbacterium, Staphylothermus, Gluconobacter, and Enterococcus were dominant at all time points. Functional genes related to petroleum degradation decreased 1~;;2 orders of magnitude. Most of the key functional genes were still detected after ozonation, allowing a rapid recovery of the microbial community after ozonation. While ozone had a large impact on the

Left-right asymmetries in the epithalamic region of the brain are widespread across vertebrates, but their magnitude and laterality varies among species. Whether these differences reflect independent origins of forebrain asymmetries or taxa-specific diversifications of an ancient vertebrate feature remains unknown. Here we show that the catshark Scyliorhinus canicula and the lampreys Petromyzon marinus and Lampetra planeri exhibit conserved molecular asymmetries between the left and right developing habenulae. Long-term pharmacological treatments in these species show that nodal signalling is essential to their generation, rather than their directionality as in teleosts. Moreover, in contrast to zebrafish, habenular left-right differences are observed in the absence of overt asymmetry of the adjacent pineal field. These data support an ancient origin of epithalamic asymmetry, and suggest that a nodal-dependent asymmetry programme operated in the forebrain of ancestral vertebrates before evolving into a variable trait in bony fish.

Podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant oceanic photosynthetic microorganism. Single-particle cryo-electron microscopy yields icosahedral and asymmetrical structures of infectious P-SSP7 with 4.6-A and 9-A resolution, respectively. The asymmetric reconstruction reveals how symmetry mismatches are accommodated among five of the gene products at the portal vertex. Reconstructions of infectious and empty particles show a conformational change of the 'valve' density in the nozzle, an orientation difference in the tail fibers, a disordering of the C terminus of the portal protein and the disappearance of the core proteins. In addition, cryo-electron tomography of P-SSP7 infecting Prochlorococcus showed the same tail-fiber conformation as that in empty particles. Our observations suggest a mechanism whereby, upon binding to the host cell, the tail fibers induce a cascade of structural alterations of the portal vertex complex that triggers DNA release.

The genes of ectoine biosynthesis pathway were identified in six species of aerobic, slightly halophilic bacteria utilizing methane, methanol or methylamine. Two types of ectoine gene cluster organization were revealed in the methylotrophs. The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA), DABA aminotransferase (EctB) and ectoine synthase (EctC) was found in methanotrophs Methylobacter marinus 7C and Methylomicrobium kenyense AMO1(T). In methanotroph Methylomicrobium alcaliphilum ML1, methanol-utilizers Methylophaga thalassica 33146(T) , Methylophaga alcalica M8 and methylamine-utilizer Methylarcula marina h1(T), the genes forming the ectABC-ask operon are preceded by ectR, encoding a putative transcriptional regulatory protein EctR. Phylogenetic relationships of the Ect proteins do not correlate with phylogenetic affiliation of the strains, thus implying that the ability of methylotrophs to produce ectoine is most likely the result of a horizontal transfer event.

Ergasilus megaceros (Copepoda: Ergasilidae) was recovered from the nasal fossae (lamellae) of the olfactory sac in 1 (1.8%) of 56 sea lampreys, Petromyzon marinus Linne, 1758, collected in May 2002 from the Cheboygan River, Michigan. Although the sea lamprey is a new host record for E. megaceros, this fish species may not be a preferred host because of its low prevalence. Ergasilus megaceros is the second ergasilid species reported from the sea lamprey in North America. This is the third report of an ergasilid species infecting the nasal fossae of fishes in North America, with E. rhinos being the only other species reported from this site.

Biological macromolecules are utilized in low-temperature synthetic methods to exert precise control over nanoparticle nucleation and placement. They enable low-temperature formation of a variety of functional nanostructured materials with properties often not achieved via conventional synthetic techniques. Here we report on the in situ visualization of a novel acidic bacterial recombinant protein, MamC, commonly present in the magnetosome membrane of several magnetotactic bacteria, including Magnetococcus marinus , strain MC-1. Our findings provide an insight into the self-assembly of MamC and point to formation of the extended protein surface, which is assumed to play an important role in the formationmore » of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.« less

The life history of the landlocked sea lamprey, Petromyzon marinus, has been described by several authors, the two most recent of which are Applegate and Wigley. The only information on the production of larvae from nests of the sea lamprey was reported by Applegate, who counted the larvae from three nests in the Ocqueoc River, a tributary of Lake Huron. The present report presents data on the hatching success of sea lamprey larvae from 19 nests in two small tributaries of southern Lake Superior and indicates greater production per nest than that recorded by Applegate. Studies were conducted by personnel of the U.S. Bureau of Commercial Fisheries on the Little Garlic River, Marquette County, Michigan, and on the Traverse River, Keweenaw County, Michigan.

A novel system combining a trap and pulsed direct current electricity was able to catch up to 75% of tagged invasive sea lamprey Petromyzon marinus in free-flowing streams. Non-target mortality was rare and impacts to non-target migration were minimal; likely because pulsed direct current only needed to be activated at night (7 hours of each day). The system was completely portable and the annual cost of the trapping system was low ($4,800 U.S. dollars). Use of the technology is poised to substantially advance integrated control of sea lamprey, which threaten a fishery valued at 7 billion U.S. dollars annually, and help restore sea lamprey populations in Europe where they are native, but imperiled. The system may be broadly applicable to controlling invasive fishes and restoring valued fishes worldwide, thus having far reaching effects on ecosystems and societies.

A novel system combining a trap and pulsed direct current electricity was able to catch up to 75% of tagged invasive sea lamprey Petromyzon marinus in free-flowing streams. Non-target mortality was rare and impacts to non-target migration were minimal; likely because pulsed direct current only needed to be activated at night (7 hours of each day). The system was completely portable and the annual cost of the trapping system was low ($4,800 U.S. dollars). Use of the technology is poised to substantially advance integrated control of sea lamprey, which threaten a fishery valued at 7 billion U.S. dollars annually, and help restore sea lamprey populations in Europe where they are native, but imperiled. The system may be broadly applicable to controlling invasive fishes and restoring valued fishes worldwide, thus having far reaching effects on ecosystems and societies.

A novel system combining a trap and pulsed direct current electricity was able to catch up to 75% of tagged invasive sea lamprey Petromyzon marinus in free-flowing streams. Non-target mortality was rare and impacts to non-target migration were minimal; likely because pulsed direct current only needed to be activated at night (7 hours of each day). The system was completely portable and the annual cost of the trapping system was low ($4,800 U.S. dollars). Use of the technology is poised to substantially advance integrated control of sea lamprey, which threaten a fishery valued at 7 billion U.S. dollars annually, and help restore sea lamprey populations in Europe where they are native, but imperiled. The system may be broadly applicable to controlling invasive fishes and restoring valued fishes worldwide, thus having far reaching effects on ecosystems and societies. PMID:27341485

In this study total of 266 fecal samples from 242 animals belonging to 113 species kept in the Poznan Zoological Garden were examined for Giardia. The cysts of Giardia were found only in five samples of feces collected from a giant toad (Bufo marinus), tamandua (Tamandua tetradactyla) and three individuals of cactus mouse (Peromyscus eremicus). Fragments of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes were successfully amplified only from the Giardia isolate obtained from the tamandua. Sequence and phylogenetic analyses showed that the Giardia isolate from the tamandua belonged to the B assemblage and showed homologies of 99% to 100% at bg, gdh and tpi loci of the same markers of parasites isolated from humans and animals in various parts of the world. This is the first molecular characterization of G. duodenalis from tamandua.

In this study, oil was applied to naturally incubated great black-backed gull (Larus marinus) and herring gull (L. argentatus) eggs, and its effects on reproductive success were assessed. Embryo survival was inversely proportional to the quantity of petroleum applied to eggshell surfaces. Dose responses, however, were dependent on embryonic age at the time of treatment. Eggs of either species, treated with 10-20 mu l of No. 2 fuel oil 4-8 days after laying, experienced significant reductions in hatching success. Embryos oiled past the midpoint of the 28-day incubation period were insensitive to as much as 100 mu l of petroleum. Fuel oil weathered outdoors for several weeks was as toxic as fresh oil to larid embryos. Only under severe conditions (e.g., large doses of petroleum contaminating young embryos) could egg oiling have a significant impact upon populations of the herring gull and species with similar life-history characteristics.

Growth was studied of five year classes of the northern brook lamprey, Ichthyomyzon fossor, collected from the Sturgeon River during intervals between treatment of the stream with a lampricide. Growth varied considerably among year classes. Larvae of the 1963 year class were slightly longer at age II and 30% longer at age III than the III-group larvae of the 1960 year class. About 6% of 558 III-group lampreys of the 1963 year class had metamorphosed by 17 August 1966. Although the sex ratio of larvae was about 1:1, 97% of the metamorphosed lampreys were males. The distribution of pigmentation on the caudal fin and upper lip in ammocoetes less than 40 mm long permitted accurate and rapid separation of northern brook lampreys from the sea lamprey, Petromyzon marinus.

Rapid environmental change due to human activities has increased rates of extinction, but some species may be able to adapt rapidly enough to deal with such changes. Our studies of feeding behaviour and physiological resistance to toxins reveal surprisingly rapid adaptive responses in Australian black snakes (Pseudechis porphyriacus) following the invasion of a lethally toxic prey item, the cane toad (Bufo marinus). Snakes from toad-exposed localities showed increased resistance to toad toxin and a decreased preference for toads as prey. Separate laboratory experiments suggest that these changes are not attributable to learning (we were unable to teach naive snakes to avoid toxic prey) or to acquired resistance (repeated sub-lethal doses did not enhance resistance). These results strongly suggest that black snake behaviour and physiology have evolved in response to the presence of toads, and have done so rapidly. Toads were brought to Australia in 1935, so these evolved responses have occurred in fewer than 23 snake generations.

High-molecular-weight materials associated with the extracellular matrix and film found on titanium and aluminum surfaces after exposure to flowing coastal seawater were isolated. This material was purified by hydroxylapatite chromatography and subsequently employed to produce antibodies in the toad, Bufo marinus. The antibodies were immobilized on a solid support and employed to isolate adhesion-enhancing, high-molecular-weight materials from the laboratory culture media of bacterial strains recovered from the respective metallic surfaces during the course of their exposure to seawater. The adhesion-enhancing materials produced by the surface-associated bacterial strains were immunologically related to the extracellular biofouling matrix material found on the surfaces from which these bacteria were isolated. The surface selectivity of these bacterial strains appeared to be based on the specificity of the interaction between adhesion-enhancing macromolecules produced by these bacteria and the surfaces in question. PMID:16346622

The lake trout (Salvelinus namaycush) stock at Gull Island Shoal in western Lake Superior was one of only a few stocks of lean lake trout in the Great Lakes that survived overfishing and predation by the sea lamprey (Petromyzon marinus). Since the mid 1960s, the abundance of wild recruits measured at age 0 and the number of age-7 to -11 wild fish recruited to the fishable stock have increased. We used the Varley-Gradwell method to test for density-dependent survival between these life stages. Survival from age-0 to ages 7–11 was not affected by increasing density, which suggests that further increases in recruitment and stock size are still possible. We suggest that testing for the existence of density-dependent survival can be used to indicate when lake trout populations are rehabilitated.

The experimental control of the sea lamprey (Petromyzon marinus) in the Great Lakes has required the collection of thousands of lampreys. Representatives of each life stage of the four species of the Lake Superior basin were examined for structural abnormalities. The most common aberration was the presence of additional tails. The accessory tails were always postanal and smaller than the normal tail. The point of origin varied; the extra tails occurred on dorsal, ventral, or lateral surfaces. Some of the extra tails were misshaped and curled, but others were normal in shape and pigment pattern. Other abnormalities in larval sea lampreys were malformed or twisted tails and bodies. The cause of the structural abnormalities is unknown. The presence of extra caudal fins could be genetically controlled, or be due to partial amputation or injury followed by abnormal regeneration. Few if any lampreys with structural abnormalities live to sexual maturity.

Dorsal and pectoral fin spines from two species of sea catfishes (Bagre marinus and Ariopsis felis) landed at 54 sites in Galveston Bay, Texas, and its sub-bays from June to October 2005 were screened with traditional cultivation-based assays and quantitative PCR assays for Vibrio vulnificus and Vibrio parahaemolyticus. V. vulnificus was present on 51.2% of fish (n = 247), with an average of 403 ± 337 SD cells g(-1). V. parahaemolyticus was present on 94.2% (n = 247); 12.8% tested positive for the virulence-conferring tdh gene, having an average 2,039 ± 2,171 SD cells g(-1). The increasing trend in seafood consumption of "trash fishes" from lower trophic levels, such as sea catfishes, warrants evaluation of their life histories for association with pathogens of concern for human consumption.

Patch-clamp studies were performed on the isolated dorsal sensory cells of the spinal cords of three species of lamprey, Ichthyomyzon unicuspis, Petromyzon marinus, and Lampetra fluviatilis, to measure changes in the amplitudes of calcium current induced by GABA and its specific antagonists and agonists. The experiments showed that GABA (4 mM) reduced the peak amplitude of the calcium current by 28.5 +/- 4.9%, with subsequent recovery to 96.2 +/- 9.2% of control (n = 45). The GABAB agonist baclofen had similar effects. The GABAA agonists glycine and taurine had no effect on the Ca2+ current. The inhibitory effect of GABA was blocked by 2-hydroxysaclofen (a GABAB antagonist), but persisted in the presence of bicuculline (a GABAA antagonist). These results are evidence that the membranes of dorsal sensory cells contain GABAB receptors, which significantly increases our understanding of the mechanisms of presynaptic inhibition in the spinal cords of the cyclostomata.

The parasitic sea lamprey, Petromyzon marinus, has in recent years spread throughout Lakes Huron and Michigan and is now firmly established in these waters (Applegate, 1949, Mich. Cons., 18 (4): 13-15). Coincident with their spread, the abundance of lake trout, Salvelinus namaycush, has declined in both lakes (Hile, 1949, Trans. Amer. Fish. Soc., 76 (1946): 121-147) and the lake trout as well as other species of fishes are showing an increase in scarring from lamprey attacks. For Lake Michigan the analysis of fishermen's questionnaires gave an increase in percentage by weight of lamprey-scarred lake trout from 31 percent in 1947 to 41 percent in 1948. The sea lamprey is now spreading through Lake Superior, the last of the Great Lakes containing a large population of lake trout.

Perkinsus chesapeaki is reported from stout razor clams Tagelus plebeius in Delaware Bay, extending the known range of P. chesapeaki north of Chesapeake Bay. P. marinus, which causes dermo disease, is prevalent in cultured and wild oysters at this site, but was not detected in T. plebeius. Evidence for the presence of disseminated neoplasia, also reported from Chesapeake Bay, was equivocal. Although P. chesapeaki infections were associated with mortality events, light infection intensities and a general lack of histopathological evidence of disease limit inferences about a causal relationship. A comparison of Ray's fluid thioglycollate medium (RFTM)-based and PCR-based detection assays highlight differences in detection capabilities related to the quantity and type of tissue processed rather than assay sensitivity per se, a point that should be considered when surveying populations for disease prevalence. Investigators are further cautioned to use care when applying and interpreting diagnostic assays when used with novel species.

The fast growing oyster aquaculture industry is greatly hindered by Perkinsus marinus and Haplosporidium nelsoni which can kill up to 80% of the production. The relationship between parasites and oyster defence mechanisms is unclear. Two defence mechanisms of the Eastern Oyster (Crassostrea virginica) were quantified at the single cell level utilising flow cytometry. Phagocytosis was measured using fluorescent beads. Respiratory burst activity was quantified as the H2O2-specific increase in dichlorofluorescein-associated fluorescence upon stimulation. These two assays distinguished three populations of haemocytes (granulocytes, hyalinocytes and intermediate cells) with unique functional characteristics. Granulocytes were most active at phagocytosis and H2O2 production while hyalinocytes were relatively inactive. The intermediate cells had moderate phagocytic and respiratory burst activity. Flow cytometry can rapidly, accurately and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the bivalve immune system.

This critical review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biology and physiology. Here, we describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quantitative measurements in living systems. “If wood is rubbed with the Pulmo marinus, it will have all the appearance of being on fire; so much so, indeed, that a walking-stick, thus treated, will light the way like a torch” (translation of Pliny the Elder from John Bostock, 1855). PMID:19771335

High-molecular-weight materials associated with the extracellular matrix and film found on titanium and aluminum surfaces after exposure to flowing coastal seawater were isolated. This material was purified by hydroxylapatite chromatography and subsequently employed to produce antibodies in the toad, Bufo marinus. The antibodies were immobilized on a solid support and employed to isolate adhesion-enhancing, high-molecular-weight materials from the laboratory culture media of bacterial strains recovered from the respective metallic surfaces during the course of their exposure to seawater. The adhesion-enhancing materials produced by the surface-associated bacterial strains were immunologically related to the extracellular biofouling matrix material found on the surfaces from which these bacteria were isolated. The surface selectivity of these bacterial strains appeared to be based on the specificity of the interaction between adhesion-enhancing macromolecules produced by these bacteria and the surfaces in question. 30 references, 6 tables.

Construction of the Welland Canal enabled shipping to by-pass Niagara Falls and enter the upper Great Lakes and also eliminated the barrier to the entry to the lakes by the sea lamprey (Petromyzon marinus Linnaeus). Within forty years the commercial fisheries of the Great Lakes was almost eliminated by this parasitic cyclostome. A search for selective chemical control of the sea lamprey was undertaken in the 1950's and culminated with the discovery of TFM (3-Trifluoromethyl-4-nitrophenol). At the request of the International Great Lakes Fishery Commission, the Bureau of Sport Fisheries and Wildlife undertook to assess the hazard of TFM to the aquatic ecosystem, to humans as well as to fish and wildlife. Studies were undertaken in Bureau laboratories as well as by contracts with university and private laboratories. Results of these studies to-date indicate that this material is not subject to biomagnification and does not pose a hazard to man or the the environment.

Use of 3-trifluoromethyl-4-nitrophenol (TFM) is limited geographically to the Great Lakes basin where it is the principal agent used in control of the sea lamprey (Petromyzon marinus). It is clear from available data that TFM has effects on the environment, but the effects reported are transient. Individual organisms and aquatic communities return to pretreatment conditions after lampricide treatments have concluded. TFM is not persistent, is detoxified, and presents minimal long-term toxicological risk. TFM is relatively nontoxic to mammals. Treatment levels do not pose a threat to wildlife. However, TFM is an estrogen agonist and additional testing to define the nature and magnitude of this effect will likely be required. Because stream treatments are done on 3 to 5 year cycles, and exposures are limited to approximately 12 h, minimal risk to aquatic organisms is expected.

Use of 3-trifluoromethyl-4-nitrophenol (TFM) is limited geographically to the Great Lakes basin where it is the principal agent used in control of the sea lamprey (Petromyzon marinus). It is clear from available data that TFM has effects on the environment, but the effects reported are transient. Individual organisms and aquatic communities return to pretreatment conditions after lampricide treatments have concluded. TFM is not persistent, is detoxified, and presents minimal long-term toxicological risk. TFM is relatively nontoxic to mammals. Treatment levels do not pose a threat to wildlife. However, TFM is an estrogen agonist and additional testing to define the nature and magnitude of this effect will likely be required. Because stream treatments are done on 3 to 5 year cycles, and exposures are limited to approximately 12 h, minimal risk to aquatic organisms is expected.

The sea lamprey (Petromyzon marinus), a jawless parasitic eel-like fish native to the Atlantic Ocean (fig. 1), was accidentally introduced into the Great Lakes in the early 20th century through the construction of shipping canals. A member of the Petromyzonidae family, the primitive parasite has been identified as a major cause of the collapse of the Great Lakes fishery in the 1940s and 1950s. The lampricides 3-trifluoromethyl-4-nitrophenol (TFM) and 2'5-dichloro-4'-nitrosalicylanilide (niclosamide) have been used to control larval sea lampreys in tributaries of the Great Lakes since the early 1960s. The lampricide TFM is the main compound used to keep sea lamprey populations in check while niclosamide is used primarily in combination with TFM as a cost-saving measure. The addition of niclosamide at a ratio of 1% to TFM will reduce the amount of TFM required for effective treatment by about 40%.

During May 1997, specimens of 7 species of anurans, that included 5 Phrynohyas venulosa Laurenti, 5 Rana forreri Boulenger, 7 Rana vaillanti Brucchi, 6 Eleutherodactylus fitzingeri Schimdt, 4 Smilisca baudinii Duméril and Bibron, 1 Leptodactylus melanonotus, and 3 Bufo marinus Linneaus, from the Guanacaste Conservation Area, Costa Rica were examined for blood parasites. Their hematozoan fauna included intraerythrocytic and intraleukocytic icosahedral viruses, a rickettsia (Aegyptianella sp.), 2 species of Hepatozoon, Lankesterella minima, 2 unknown species of apicomplexans, 9 morphologically distinct types of trypanosomes, and 2 species of microfilariae. Rana vaillanti, the most aquatic species of frog, harbored the most species of parasites. Recent evidence indicates that morphological changes in the highly pleomorphic trypanosomes of anurans from different geographical regions have not kept pace with biochemical (isozyme) and molecular (DNA sequence) changes. Describing new species based solely on bloodstream trypomastigotes is discouraged. Additional criteria described herein should be applied when naming new species of anuran trypanosomes.

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry. PMID:24489862

Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world's oceans.

Male spawning-run sea lampreys Petromyzon marinus were exposed to various doses of cobalt-60 or cesium-137 radiation in an attempt to sterilize them for use in a program for controlling sea lampreys through the release of sterile males. Males captured and irradiated during the early part of the upstream migration were not effectively sterilized at the doses tested. After irradiation, the sea lampreys were more susceptible to fungal infections by Saprolegnia sp., and many died without attempting to spawn. Males captured and irradiated during the middle and late parts of the spawning migration were effectively sterilized at a dose of 2,000 rads. However, some radiation-induced mortality was observed in males captured and irradiated during the middle part of the spawning migration. Radiation is not as effective as the chemosterilant bisazir for sterilizing male sea lampreys.

Homeobox genes represent a class of transcription factors that play key roles in the regulation of embryogenesis and development. Here we report the identification of a homeobox-containing gene family that is highly conserved at both the nucleotide and amino acid levels in a diverse number of species. These species encompass both vertebrate and invertebrate phylogenies, ranging from Homo sapiens to Drosophila melanogaster. In humans, at least two homeobox sequences from this family were identified representing a previously reported member of this family as well as a novel homeobox sequence that we physically mapped to the 10q25.2-q26.3 region of human Chromosome (Chr) 10. Multiple members of this family were also detected in three additional vertebrate species including Equus caballus (horse), Gallus gallus (Chicken), and Mus musculus (mouse), whereas only single members were detected in Tripneustes gratilla (sea urchin), Petromyzon marinus (lamprey), Salmo salar (salmon), Ovis aries (sheep), and D. melanogaster (fruit fly).

We describe a water-recirculating system for inducing spawning of sea lampreys (Petromyzon marinus) held under laboratory conditions. Water temperature in the system was gradually increased to and maintained at 18 ± 2°C, the optimal temperature for spawning. About 10% freshwater was added daily to prevent buildup of waste products. Sea lampreys were provided substrate (approximately 3–6 cm in diameter) to build nests, and a water velocity of 0.2–0.3 m!s was maintained with an electric trolling motor. Sea lampreys held in this system exhibited characteristic spawning behavior. Prolarvae produced from artificial fertilization of gametes developed according to the standard timeline.

A novel system combining a trap and pulsed direct current electricity was able to catch up to 75% of tagged invasive sea lamprey Petromyzon marinus in free-flowing streams. Non-target mortality was rare and impacts to non-target migration were minimal; likely because pulsed direct current only needed to be activated at night (7 hours of each day). The system was completely portable and the annual cost of the trapping system was low ($4,800 U.S. dollars). Use of the technology is poised to substantially advance integrated control of sea lamprey, which threaten a fishery valued at 7 billion U.S. dollars annually, and help restore sea lamprey populations in Europe where they are native, but imperiled. The system may be broadly applicable to controlling invasive fishes and restoring valued fishes worldwide, thus having far reaching effects on ecosystems and societies.

Connectivity is a central issue in the development, sustainability and effectiveness of networks of Marine Protected Areas (MPAs). In populations with site attached adults, connectivity is limited to dispersal in the pelagic larval stage. While biophysical models have been widely used to infer early dispersal, empirical evidence through sources such as otolith microchemistry can provide a means of evaluating model predictions. In the present study, connectivity in the lesser sandeel, Ammodytes marinus, was investigated using LA-ICP-MS otolith microchemistry. Otoliths from juveniles (age 0) were examined from four Scottish spawning areas predicted to differ in terms of larval retention rates and connectivity based on past biophysical models. There were significant spatial differences in otolith post-settled juvenile chemistry among locations at a scale of 100-400 km. Differences in near core chemistry pointed to three chemically distinct natal sources, as identified by a cluster analysis, contributing to settlement locations.

Background Based on extensive mitochondrial DNA (mtDNA) sequence data, we previously showed that the model of speciation among species of herring gull (Larus argentatus) complex was not that of a ring species, but most likely due more complex speciation scenario's. We also found that two species, herring gull and glaucous gull (L. hyperboreus) displayed an unexpected biphyletic distribution of their mtDNA haplotypes. It was evident that mtDNA sequence data alone were far from sufficient to obtain a more accurate and detailed insight into the demographic processes that underlie speciation of this complex, and that extensive autosomal genetic analysis was warranted. Results For this reason, the present study focuses on the reconstruction of the phylogeographic history of a limited number of gull species by means of a combined approach of mtDNA sequence data and 230 autosomal amplified fragment length polymorphism (AFLP) loci. At the species level, the mtDNA and AFLP genetic data were largely congruent. Not only for argentatus and hyperboreus, but also among a third species, great black-backed gull (L. marinus) we observed two distinct groups of mtDNA sequence haplotypes. Based on the AFLP data we were also able to detect distinct genetic subgroups among the various argentatus, hyperboreus, and marinus populations, supporting our initial hypothesis that complex demographic scenario's underlie speciation in the herring gull complex. Conclusions We present evidence that for each of these three biphyletic gull species, extensive mtDNA introgression could have taken place among the various geographically distinct subpopulations, or even among current species. Moreover, based on a large number of autosomal AFLP loci, we found evidence for distinct and complex demographic scenario's for each of the three species we studied. A more refined insight into the exact phylogeographic history within the herring gull complex is still impossible, and requires detailed autosomal

The galectin CvGal1 from the eastern oyster (Crassostrea virginica), which possesses four tandemly arrayed carbohydrate recognition domains, was previously shown to display stronger binding to galactosamine and N-acetylgalactosamine relative to d-galactose. CvGal1 expressed by phagocytic cells is “hijacked” by the parasite Perkinsus marinus to enter the host, where it proliferates and causes systemic infection and death. In this study, a detailed glycan array analysis revealed that CvGal1 preferentially recognizes type 2 blood group A oligosaccharides. Homology modeling of the protein and its oligosaccharide ligands supported this preference over type 1 blood group A and B oligosaccharides. The CvGal ligand models were further validated by binding, inhibition, and competitive binding studies of CvGal1 and ABH-specific monoclonal antibodies with intact and deglycosylated glycoproteins, hemocyte extracts, and intact hemocytes and by surface plasmon resonance analysis. A parallel glycomic study carried out on oyster hemocytes (Kurz, S., Jin, C., Hykollari, A., Gregorich, D., Giomarelli, B., Vasta, G. R., Wilson, I. B. H., and Paschinger, K. (2013) J. Biol. Chem. 288,) determined the structures of oligosaccharides recognized by CvGal1. Proteomic analysis of the hemocyte glycoproteins identified β-integrin and dominin as CvGal1 “self”-ligands. Despite strong CvGal1 binding to P. marinus trophozoites, no binding of ABH blood group antibodies was observed. Thus, parasite glycans structurally distinct from the blood group A oligosaccharides on the hemocyte surface may function as potentially effective ligands for CvGal1. We hypothesize that carbohydrate-based mimicry resulting from the host/parasite co-evolution facilitates CvGal1-mediated cross-linking to β-integrin, located on the hemocyte surface, leading to cell activation, phagocytosis, and host infection. PMID:23824193

A polyphasic study was designed to determine the taxonomic provenance of three Modestobacter strains isolated from an extreme hyper-arid Atacama Desert soil. The strains, isolates KNN 45-1a, KNN 45-2b(T) and KNN 45-3b, were shown to have chemotaxonomic and morphological properties in line with their classification in the genus Modestobacter. The isolates had identical 16S rRNA gene sequences and formed a branch in the Modestobacter gene tree that was most closely related to the type strain of Modestobacter marinus (99.6% similarity). All three isolates were distinguished readily from Modestobacter type strains by a broad range of phenotypic properties, by qualitative and quantitative differences in fatty acid profiles and by BOX fingerprint patterns. The whole genome sequence of isolate KNN 45-2b(T) showed 89.3% average nucleotide identity, 90.1% (SD: 10.97%) average amino acid identity and a digital DNA-DNA hybridization value of 42.4±3.1 against the genome sequence of M. marinus DSM 45201(T), values consistent with its assignment to a separate species. On the basis of all of these data, it is proposed that the isolates be assigned to the genus Modestobacter as Modestobacter caceresii sp. nov. with isolate KNN 45-2b(T) (CECT 9023(T)=DSM 101691(T)) as the type strain. Analysis of the whole-genome sequence of M. caceresii KNN 45-2b(T), with 4683 open reading frames and a genome size of ∽4.96Mb, revealed the presence of genes and gene-clusters that encode for properties relevant to its adaptability to harsh environmental conditions prevalent in extreme hyper arid Atacama Desert soils.

Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in

Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes.

Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes. PMID:25482621

A hydrodynamic-oyster population model was developed to assess the effect of changes in freshwater inflow on oyster populations in Galveston Bay, Texas, USA. The population model includes the effects of environmental conditions, predators, and the oyster parasite, Perkinsus marinus, on oyster populations. The hydrodynamic model includes the effects of wind stress, river runoff, tides, and oceanic exchange on the circulation of the bay. Simulations were run for low, mean, and high freshwater inflow conditions under the present (1993) hydrology and predicted hydrologies for 2024 and 2049 that include both changes in total freshwater inflow and diversions of freshwater from one primary drainage basin to another. Freshwater diversion to supply the Houston metropolitan area is predicted to negatively impact oyster production in Galveston Bay. Fecundity and larval survivorship both decline. Mortality from Perkinsus marinus increases, but to a lesser extent. A larger negative impact in 2049 relative to 2024 originates from the larger drop in fecundity under that hydrology. Changes in recruitment and mortality, resulting in lowered oyster abundance, occur because the bay volume available for mixing freshwater input from the San Jacinto and Buffalo Bayou drainage basins that drain metropolitan Houston is small in comparison to the volume of Trinity Bay that presently receives the bulk of the bay's freshwater inflow. A smaller volume for mixing results in salinities that decline more rapidly and to a greater extent under conditions of high freshwater discharge.Thus, the decline in oyster abundance results from a disequilibrium between geography and salinity brought about by freshwater diversion. Although the bay hydrology shifts, available hard substrate does not. The simulations stress the fact that it is not just the well-appreciated reduction in freshwater inflow that can result in decreased oyster production. Changing the location of freshwater inflow can also

Percent mortality of lake trout Salvelinus namaycush subjected to single sea lamprey Petromyzon marinus attacks did not differ significantly between lower-temperature (mortality = 54%; temperature [le] 10[degrees]C; N = 33) and higher-temperature (mortality = 69%; temperature = 12.8-14.4[degrees]C; N = 45) laboratory studies conducted from 1 June to 28 November 1989. However, sea lampreys fed longer and killed fewer fish in colder water (mean attachment 467.0 h; 18 fish killed) than in warmer water (mean attachment 161.7 h; 31 fish killed), probably because food consumption was lower in colder water. These results indicate that the number of fish killed by sea lampreys could be much greater in warmer water and that temperature must be considered when fish losses from sea lamprey attacks are estimated. Previous studies (Swink and Hanson 1989; Swink 1990) of the effects of single sea lamprey Petromyzon marinus attacks on lake trout Salvelinus namaycush showed significantly less lake trout mortality at temperatures of 10[degrees]C and lower than at higher temperatures. The reduced host mortality, however, could not be attributed solely to lower temperature because warmwater and coldwater attacks occurred during different seasons. In those studies, the author was unable to hold water temperature at 10[degrees]C or less in late summer and early fall, when most fish are killed by sea lampreys in the Great Lakes (Christie and Kolenosky 1980; Bergstedt and Schneider 1988). Modifications to the fish holding facilities at the Hammond Bay Biological Station in 1988 allowed maintenance of a limited amount of water at 10[degrees]C or less throughout the year. Hence, the objective of this study was to compare sea lamprey-induced mortality of lake trout at 10[degrees]C or less with that at 12.8-14.4[degrees]C during the normal feeding season (June through November). 15 refs., 1 fig., 1 tab.

Urea is actively transported inwardly (J{sub i}) across the skin of the green toad Bufo viridis. J{sub i} is markedly enhanced in toads adapted to hypertonic saline. The authors studied urea transport across the skin of Bufo viridis under a variety of experimental conditions, including treatment with amiloride and phloretin, agents that inhibit urea permeability in the bladder of Bufo marinus. Amiloride (10{sup {minus}4} M) significantly inhibited J{sub i} in both adapted and unadapted animals and was unaffected by removal of sodium from the external medium. Phloretin (10{sup {minus}4} M) significantly inhibited J{sub i} in adapted animals by 23-46%; there was also a reduction in J{sub i} in unadapted toads at 10{sup {minus}4} and 5 {times} 10{sup {minus}4} M phloretin. A dose-response study revealed that the concentration of phloretin causing half-maximal inhibition (K{sub {1/2}}) was 5 {times} 10{sup {minus}4} M for adapted animals. J{sub i} was unaffected by the substitution of sucrose for Ringer solution or by ouabain. They conclude (1) the process of adaptation appears to involve an increase in the number of amiloride- and phloretin-inhibitable urea transport sites in the skin, with a possible increase in the affinity of the sites for phloretin; (2) the adapted skin resembles the Bufo marinus urinary bladder with respect to amiloride and phloretin-inhibitable sites; (3) they confirm earlier observations that J{sub i} is independent of sodium transport.

Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in

Background and Objective Aggressive periodontitis (AgP) is prevalent and shows a rapid course in African individuals. Although a strong focus has been placed on Aggregatibacter actinomycetemcomitans, new methods support the existence of a complex subgingival microflora in AgP. The purpose of the present study was to map the subgingival microbiota as well as explore the presence of A. actinomycetemcomitans and the JP2 clone in a group of Sudanese individuals with AgP, using different analytical methods. Material and Methods A study population consisting of 19 patients with AgP was recruited from patients seeking treatment at University of Science and Technology (UST) in Khartoum. Fifteen healthy subjects were included as controls. Plaque samples were analyzed for 272 taxa using human oral microbe identification microarrays and for 26 periodontal taxa using DNA-DNA hybridization checkerboard. Conventional polymerase chain reaction (PCR) was applied for the detection of A. actinomycetemcomitans and the JP2 clone in plaque. Saliva from patients with AgP was analyzed using quantitative PCR (qPCR) for the detection of A. actinomycetemcomitans. Results Eubacterium yurii was detected more frequently in patients with AgP than in controls, and E. nodatum was found in patients with AgP only. A. actinomycetemcomitans was found in plaque samples of two (12%) patients by human oral microbe identification microarrays and in five (29%) patients with AgP by conventional PCR, as well as in six (32%) of the AgP saliva samples by qPCR. The JP2 clone was identified in only one patient. Conclusion The classical periodontal pathogens were not present in high amounts in AgP in the population studied here. Species of Eubacterium, which are not typically associated with AgP, were often detected in individuals with disease. Using laboratory methods with different sensitivities and detection levels allowed identification of variances in microbial communities. The findings reported in this

The gastrointestinal microbiota produces short-chain fatty acids, especially butyrate, which affect colonic health, immune function and epigenetic regulation. To assess the effects of nutrition and aging on the production of butyrate, the butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium clusters lV and XlVa, the main butyrate producers, were analysed. Faecal samples of young healthy omnivores (24 ± 2.5 years), vegetarians (26 ± 5 years) and elderly (86 ± 8 years) omnivores were evaluated. Diet and lifestyle were assessed in questionnaire-based interviews. The elderly had significantly fewer copies of the butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014), while vegetarians showed the highest number of copies (P=0.048). The thermal denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting curve related to Roseburia/Eubacterium rectale spp. was significantly more variable in the vegetarians than in the elderly. The Clostridium cluster XIVa was more abundant in vegetarians (P=0.049) and in omnivores (P<0.01) than in the elderly group. Gastrointestinal microbiota of the elderly is characterized by decreased butyrate production capacity, reflecting increased risk of degenerative diseases. These results suggest that the butyryl-CoA:acetate CoA-transferase gene is a valuable marker for gastrointestinal microbiota function.

Epulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2,000-fold in volume), and undergoes a complex daily life cycle. In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm. Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Fluorescence measurements of DNA, RNA, and other cell components indicate the following. DNA quantity is proportional to cell volume over cell lengths of ∼30 μm to >500 μm. For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA. And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size. The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and “pinching” of DNA near the middle of the cell in the absence of new wall formation. Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines. PMID:9791108

Patients with acquired immunodeficiency syndrome (AIDS) are at high risk of opportunistic infections. Oral manifestations have been associated with the level of immunosuppression, these include periodontal diseases, and understanding the microbial populations in the oral cavity is crucial for clinical management. The aim of this study was to examine the salivary bacterial diversity in patients newly admitted to the AIDS ward of the Public Health Clinical Center (China). Saliva samples were collected from 15 patients with AIDS who were randomly recruited between December 2013 and March 2014. Extracted DNA was used as template to amplify bacterial 16S rRNA. Sequencing of the amplicon library was performed using a 454 GS-FLX Titanium sequencing platform. Reads were optimized and clustered into operational taxonomic units for further analysis. A total of 10 bacterial phyla (106 genera) were detected. Firmicutes, Bacteroidetes, and Proteobacteria were preponderant in the salivary microbiota in AIDS patients. The pathogen, Capnocytophaga sp., and others not considered pathogenic such as Neisseria elongata, Streptococcus mitis, and Mycoplasma salivarium but which may be opportunistic infective agents were detected. Dialister pneumosintes, Eubacterium infirmum, Rothia mucilaginosa, and Treponema parvum were preponderant in AIDS patients with periodontitis. Patients with necrotic periodontitis had a distinct salivary bacterial profile from those with chronic periodontitis. This is the first study using advanced sequencing techniques focused on hospitalized AIDS patients showing the diversity of their salivary microbiota. PMID:26191508

Dietary modulation of the gut microbiota impacts human health. Here we investigated the hitherto unknown effects of resistant starch type 4 (RS4) enriched diet on gut microbiota composition and short-chain fatty acid (SCFA) concentrations in parallel with host immunometabolic functions in twenty individuals with signs of metabolic syndrome (MetS). Cholesterols, fasting glucose, glycosylated haemoglobin, and proinflammatory markers in the blood as well as waist circumference and % body fat were lower post intervention in the RS4 group compared with the control group. 16S-rRNA gene sequencing revealed a differential abundance of 71 bacterial operational taxonomic units, including the enrichment of three Bacteroides species and one each of Parabacteroides, Oscillospira, Blautia, Ruminococcus, Eubacterium, and Christensenella species in the RS4 group. Gas chromatography-mass spectrometry revealed higher faecal SCFAs, including butyrate, propionate, valerate, isovalerate, and hexanoate after RS4-intake. Bivariate analyses showed RS4-specific associations of the gut microbiota with the host metabolic functions and SCFA levels. Here we show that dietary RS4 induced changes in the gut microbiota are linked to its biological activity in individuals with signs of MetS. These findings have potential implications for dietary guidelines in metabolic health management.

Increasing evidence suggests that altered gut microbiota is implicated in the pathogenesis of hepatitis B virus-induced chronic liver disease (HBV-CLD). However, the structure and composition of the oral microbiota of patients with HBV-CLD remains unclear. High-throughput pyrosequencing showed that decreased oral bacterial diversity was found in patients with HBV-CLD. The Firmicutes/Bacteroidetes ratio was increased significantly, which indicated that dysbiosis of the oral microbiota participated in the process of HBV-CLD development. However, the changing patterns of the oral microbiota in patients with HBV-induced liver cirrhosis (LC) were almost similar to patients with chronic hepatitis B (CHB). HBV infection resulted in an increase in potential H2S- and CH3SH-producing phylotypes such as Fusobacterium, Filifactor, Eubacterium, Parvimonas and Treponema, which might contribute to the increased oral malodor. These key oral-derived phylotypes might invade into the gut as opportunistic pathogens and contribute to altering the composition of the gut microbiota. This study provided important clues that dysbiosis of the oral microbiota might be involved in the development of HBV-CLD. Greater understanding of the relationships between the dysbiosis of oral microbiota and the development of HBV-CLD might facilitate the development of non-invasive differential diagnostic procedures and targeted treatments of HBV-CLD patients harbouring specific oral phylotypes.

Long-term diet influences the structure and activity of the trillions of microorganisms residing in the human gut1–5, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here, we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila, and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale, and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals2, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi, and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids, and the outgrowth of microorganisms capable of triggering inflammatory bowel disease6. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles. PMID:24336217

Emerging evidence has suggested a potential impact of gut microbiota on the pathophysiology of heart failure (HF). However, it is still unknown whether HF is associated with dysbiosis in gut microbiota. We investigated the composition of gut microbiota in patients with HF to elucidate whether gut microbial dysbiosis is associated with HF. We performed 16S ribosomal RNA gene sequencing of fecal samples obtained from 12 HF patients and 12 age-matched healthy control (HC) subjects, and analyzed the differences in gut microbiota. We further compared the composition of gut microbiota of 12 HF patients younger than 60 years of age with that of 10 HF patients 60 years of age or older. The composition of gut microbial communities of HF patients was distinct from that of HC subjects in both unweighted and weighted UniFrac analyses. Eubacterium rectale and Dorea longicatena were less abundant in the gut microbiota of HF patients than in that of HC subjects. Compared to younger HF patients, older HF patients had diminished proportions of Bacteroidetes and larger quantities of Proteobacteria. The genus Faecalibacterium was depleted, while Lactobacillus was enriched in the gut microbiota of older HF patients. These results suggest that patients with HF harbor significantly altered gut microbiota, which varies further according to age. New concept of heart-gut axis has a great potential for breakthroughs in the development of novel diagnostic and therapeutic approach for HF.

OBJECTIVE: Changes in the neonatal gut environment allow for the colonization of the mucin layer and lumen by anaerobic bacteria. The aim of the present study was to evaluate Bifidobacterium, Lactobacillus and Lactococcus colonization through the first year of life in a group of 12 Brazilian infants and to correlate these data with the levels of Escherichia coli. The presence of anaerobic members of the adult intestinal microbiota, including Eubacterium limosum and Faecalibacterium prausnitzii, was also evaluated. METHODS: Fecal samples were collected during the first year of life, and 16S rRNA from anaerobic and facultative bacteria was detected by real-time PCR. RESULTS: Bifidobacterium was present at the highest levels at all of the studied time points, followed by E. coli and Lactobacillus. E. limosum was rarely detected, and F. prausnitzii was detected only in the samples from the latest time points. CONCLUSION: These results are consistent with reports throughout the world on the community structure of the intestinal microbiota in infants fed a milk diet. Our findings also provide evidence for the influence of the environment on intestinal colonization due to the high abundance of E. coli. The presence of important anaerobic genera was observed in Brazilian infants living at a low socioeconomic level, a result that has already been well established for infants living in developed countries. PMID:28355361

The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 {angstrom}, {beta} = 104.9{sup o}; the crystal diffracted to a resolution of 1.9 {angstrom}. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 {angstrom}, and diffracted to 1.75 {angstrom} resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2{sub 1} and R32, respectively.

Tetrahydrofolate (THF), a biologically active form of the vitamin folate (B{sub 9}), is an essential cofactor in one-carbon transfer reactions. In bacteria, expression of folate-related genes is controlled by feedback modulation in response to specific binding of THF and related compounds to a riboswitch. Here, we present the X-ray structures of the THF-sensing domain from the Eubacterium siraeum riboswitch in the ligand-bound and unbound states. The structure reveals an 'inverted' three-way junctional architecture, most unusual for riboswitches, with the junction located far from the regulatory helix P1 and not directly participating in helix P1 formation. Instead, the three-way junction, stabilized by binding to the ligand, aligns the riboswitch stems for long-range tertiary pseudoknot interactions that contribute to the organization of helix P1 and therefore stipulate the regulatory response of the riboswitch. The pterin moiety of the ligand docks in a semiopen pocket adjacent to the junction, where it forms specific hydrogen bonds with two moderately conserved pyrimidines. The aminobenzoate moiety stacks on a guanine base, whereas the glutamate moiety does not appear to make strong interactions with the RNA. In contrast to other riboswitches, these findings demonstrate that the THF riboswitch uses a limited number of available determinants for ligand recognition. Given that modern antibiotics target folate metabolism, the THF riboswitch structure provides insights on mechanistic aspects of riboswitch function and may help in manipulating THF levels in pathogenic bacteria

The cabbage butterfly, Pieris rapae, and related species possess a previously unknown ADP-ribosylating toxin, guanine specific ADP-ribosyltransferase. This enzyme toxin, known as pierisin, consists of enzymatic N-terminal domain and receptor-binding C-terminal domain, or typical AB-toxin structure. Pierisin efficiently transfers an ADP-ribosyl moiety to the N(2) position of the guanine base of dsDNA. Receptors for pierisin are suggested to be the neutral glycosphingolipids, globotriaosylceramide (Gb3), and globotetraosylceramide (Gb4). This DNA-modifying toxin exhibits strong cytotoxicity and induces apoptosis in various human cell lines, which can be blocked by Bcl-2. Pierisin also produces detrimental effects on the eggs and larvae of the non-habitual parasitoids. In contrast, a natural parasitoid of the cabbage butterfly, Cotesia glomerata, was resistant to this toxin. The physiological role of pierisin in the butterfly is suggested to be a defense factor against parasitization by wasps. Other type of DNA ADP-ribosyltransferase is present in certain kinds of edible clams. For example, the CARP-1 protein found in Meretrix lamarckii consists of an enzymatic domain without a possible receptor-binding domain. Pierisin and CARP-1 are almost fully non-homologous at the amino acid sequence level, but other ADP-ribosyltransferases homologous to pierisin are present in different biological species such as eubacterium Streptomyces. Possible diverse physiological roles of the DNA ADP-ribosyltransferases are discussed.

The "in vitro" susceptibility to roxithromycin and three other macrolides of 236 anaerobes isolated from clinical samples in 1984/1985 was determined by an agar-dilution method on Wilkins Chalgren medium. 90% of Gram positive cocci were susceptible to both roxithromycin and josamycin (MIC less than 1 mg/l, whereas 1 mg/l erythromycin and 2 mg/l spiramycin were able to inhibit respectively 46 and 86% of the same tested strains. No resistance to the four macrolides was observed among Eubacterium, propionibacterium and Bifidobacterium. Two C. perfringens strains and one C. difficile strain were resistant to all four macrolides, while 97% of Clostridium sp. strains were inhibited by 4 mg/l erythromycin, josamycin or roxithromycin. Against Gram positive anaerobes, roxithromycin was equal or superior to erythromycin and spiramycin. At a concentration of 4 mg/l, roxithromycin inhibited 82% of B. fragilis strains. Roxithromycin and josamycin were more active against Gram negative bacilli that erythromycin and spiramycin. Macrolides had no effect on Fusobacterium strains. In this study, 4 mg/l roxithromycin inhibited 217 of the 236 anaerobic strains investigated (92%).

A round-spore-forming Bacillus species that produces an exosporium was isolated from the surface of the Mars Odyssey spacecraft. This novel species has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus and is a Gram-positive, aerobic, rod-shaped, endospore-forming eubacterium. Ultrathin sections of the spores showed the presence of an exosporium, spore coat, cortex and core. 16S rDNA sequence similarities between this strain, Bacillus fusiformis and Bacillus silvestris were approximately 96% and DNA-DNA reassociation values with these two bacilli were 23 and 17%, respectively. Spores of the novel species were resistant to desiccation, H2O2 and UV and gamma radiation. Of all strains tested, the spores of this strain were the most consistently resistant and survived all of the challenges posed, i.e. exposure to conditions of desiccation (100% survival), H2O2 (26% survival), UV radiation (10% survival at 660 J m(-2)) and gamma radiation (0.4% survival). The name proposed for this novel bacterium is Bacillus odysseyi sp. nov.; the type strain is 34hs-1T (=ATCC PTA-4993T=NRRL B-30641T=NBRC 100172T).

One of the spore-formers isolated from a spacecraft-assembly facility, belonging to the genus Bacillus, is described on the basis of phenotypic characterization, 16S rDNA sequence analysis and DNA-DNA hybridization studies. It is a Gram-positive, facultatively anaerobic, rod-shaped eubacterium that produces endospores. The spores of this novel bacterial species exhibited resistance to UV, gamma-radiation, H2O2 and desiccation. The 18S rDNA sequence analysis revealed a clear affiliation between this strain and members of the low G+C Firmicutes. High 16S rDNA sequence similarity values were found with members of the genus Bacillus and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain FO-92T and Bacillus benzoevorans DSM 5391T was very high. However, molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus, but DNA-DNA hybridization data support the proposal of FO-92T as Bacillus nealsonii sp. nov. (type strain is FO-92T =ATCC BAAM-519T =DSM 15077T).

The predominant ureolytic bacteria in the pig large intestine were determined while growing pigs were fed a basal diet or basal diet plus copper sulfate, Aureo SP250, or clinoptilolite. Fecal samples were collected from four pigs fed each diet at 3, 9, and 14 weeks and analyzed for total colony counts and percent ureolytic bacteria. Fecal urease activity, ammonia nitrogen, and identity of the ureolytic bacteria were determined at 14 weeks. Copper sulfate and Aureo SP250 reduced the number of ureolytic organisms, with a marked decrease occurring in the Streptococcus spp., which made up 74% of the ureolytic isolates from the pigs on the basal diet. Other ureolytic species detected at lower concentrations were Staphylococcus spp., Selenomonas ruminantium, Bacteroides multiacidus, and Eubacterium limosum. Copper sulfate also reduced fecal urease activity (P less than 0.10). Fecal ammonia concentrations were not different between pigs fed the various diets. These data suggest that the streptococci are the most numerous ureolytic species in the pig intestinal tract and are significantly reduced by copper sulfate and Aureo SP250; however, only copper sulfate reduced intestinal urease activity.

A strictly chemo-organotrophic, anaerobic bacterium was isolated from an olive mill wastewater treatment digester on syringate and designated strain SR1T. The cells were slightly curved rods, stained Gram-positive and possessed terminal spores. Strain SR1T utilized crotonate, methanol and a wide range of aromatic compounds including 3,4,5-trimethoxybenzoate (TMB), 3,4,5-trimethoxycinnamate (TMC), syringate, 3,4,5-trimethoxyphenylacetate (TMPA), 3,4,5-trimethoxyphenylpropionate (TMPP), ferulate, sinapate, vanillate, 3,4-dimethoxybenzoate, 2,3-dimethoxybenzoate, gallate, 2,4,6-trihydroxybenzoate (THB), pyrogallol, phloroglucinol and quercetin as carbon and energy sources. Acetate and butyrate were produced from aromatic compounds, methanol and crotonate whereas methanethiol (MT) was produced from methoxylated aromatic compounds and methanol. Strain SR1T had a G + C content of 38 mol% and grew optimally between 37 and 40 degrees C at pH 7.2 on a crotonate-containing medium. Phylogenetically, strain SR1T was a member of cluster XIVa of the Clostridiales group and shared a sequence similarity of 90% with Clostridum aminovalericum and Eubacterium fissicatena. Consequently, its precise neighbourliness to any one of them depended on the selection of strains of the cluster. On the basis of the phylogenetic and phenotypic evidence presented in this paper, the designation of strain SR1T as Sporobacterium olearium gen. nov., sp. nov. is proposed. The type strain is SR1T (= DSM 12504T).

Fecal samples were obtained from a cohort of 330 healthy Danish infants at 9, 18, and 36 months after birth, enabling characterization of interbacterial relationships by use of quantitative PCR targeting 31 selected bacterial 16S rRNA gene targets representing different phylogenetic levels. Nutritional parameters and measures of growth and body composition were determined and investigated in relation to the observed development in microbiota composition. We found that significant changes in the gut microbiota occurred, particularly from age 9 to 18 months, when cessation of breastfeeding and introduction of a complementary feeding induce replacement of a microbiota characterized by lactobacilli, bifidobacteria, and Enterobacteriaceae with a microbiota dominated by Clostridium spp. and Bacteroides spp. Classification of samples by a proxy enterotype based on the relative levels of Bacteroides spp. and Prevotella spp. showed that enterotype establishment occurs between 9 and 36 months. Thirty percent of the individuals shifted enterotype between 18 and 36 months. The composition of the microbiota was most pronouncedly influenced by the time of cessation of breastfeeding. From 9 to 18 months, a positive correlation was observed between the increase in body mass index and the increase of the short-chain-fatty-acid-producing clostridia, the Clostridum leptum group, and Eubacterium hallii. Considering previously established positive associations between rapid infant weight gain, early breastfeeding discontinuation, and later-life obesity, the corresponding microbial findings seen here warrant attention. PMID:24584251

Fecal samples were obtained from a cohort of 330 healthy Danish infants at 9, 18, and 36 months after birth, enabling characterization of interbacterial relationships by use of quantitative PCR targeting 31 selected bacterial 16S rRNA gene targets representing different phylogenetic levels. Nutritional parameters and measures of growth and body composition were determined and investigated in relation to the observed development in microbiota composition. We found that significant changes in the gut microbiota occurred, particularly from age 9 to 18 months, when cessation of breastfeeding and introduction of a complementary feeding induce replacement of a microbiota characterized by lactobacilli, bifidobacteria, and Enterobacteriaceae with a microbiota dominated by Clostridium spp. and Bacteroides spp. Classification of samples by a proxy enterotype based on the relative levels of Bacteroides spp. and Prevotella spp. showed that enterotype establishment occurs between 9 and 36 months. Thirty percent of the individuals shifted enterotype between 18 and 36 months. The composition of the microbiota was most pronouncedly influenced by the time of cessation of breastfeeding. From 9 to 18 months, a positive correlation was observed between the increase in body mass index and the increase of the short-chain-fatty-acid-producing clostridia, the Clostridum leptum group, and Eubacterium hallii. Considering previously established positive associations between rapid infant weight gain, early breastfeeding discontinuation, and later-life obesity, the corresponding microbial findings seen here warrant attention.

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

The presence of yeast cells could stimulate hydrogen utilization of acetogens and enhance acetogenesis. To understand the roles of acetogens in rumen fermentation, an in vitro rumen fermentation experiment was conducted with addition of acetogen strain (TWA4) and/or Saccharomyces cerevisiae fermentation product (XP). A 2×2 factorial design with two levels of TWA4 (0 or 2×107 cells/ml) and XP (0 or 2 g/L) was performed. Volatile fatty acids (VFAs) were increased (P<0.05) in XP and TWA4XP, while methane was increased only in TWA4XP (P<0.05). The increase rate of microorganisms with formyltetrahydrofolate synthetase, especially acetogens, was higher than that of methanogens under all treatments. Lachnospiraceae was predominant in all acetogen communities, but without close acetyl-CoA synthase (ACS) amino acid sequences from cultured isolates. Low-Acetitomaculum ruminis-like ACS was predominant in all acetogen communities, while four unique phylotypes in XP treatment were all amino acid identified low-Eubacterium limosum-like acetogens. It differs to XP treatment that more low-A. ruminis-like and less low-E. limosum-like sequences were identified in TWA4 and TWA4XP treatments. Enhancing acetogenesis by supplementation with an acetogen strain and/or yeast cells may be an approach to mitigate methane, by targeting proper acetogens such as uncultured low-E. limosum-like acetogens. PMID:26238546

Faecalibacterium prausnitzii is one of the most abundant commensal bacteria in the healthy human large intestine, but information on genetic diversity and substrate utilization is limited. Here, we examine the phylogeny, phenotypic characteristics, and influence of gut environmental factors on growth of F. prausnitzii strains isolated from healthy subjects. Phylogenetic analysis based on the 16S rRNA sequences indicated that the cultured strains were representative of F. prausnitzii sequences detected by direct analysis of fecal DNA and separated the available isolates into two phylogroups. Most F. prausnitzii strains tested grew well under anaerobic conditions on apple pectin. Furthermore, F. prausnitzii strains competed successfully in coculture with two other abundant pectin-utilizing species, Bacteroides thetaiotaomicron and Eubacterium eligens, with apple pectin as substrate, suggesting that this species makes a contribution to pectin fermentation in the colon. Many F. prausnitzii isolates were able to utilize uronic acids for growth, an ability previously thought to be confined to Bacteroides spp. among human colonic anaerobes. Most strains grew on N-acetylglucosamine, demonstrating an ability to utilize host-derived substrates. All strains tested were bile sensitive, showing at least 80% growth inhibition in the presence of 0.5 μg/ml bile salts, while inhibition at mildly acidic pH was strain dependent. These attributes help to explain the abundance of F. prausnitzii in the colonic community but also suggest factors in the gut environment that may limit its distribution.

Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme. PMID:8550491

A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO2 and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes. PMID:4608322

An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments. Images PMID:2705765

Quantitative and qualitative examination of anaerobically isolated flora of the gingival crevice and saliva was carried out. It was found that half the organisms were anaerobes and that there were twice as many gram-positive organisms as there were gram-negative ones. Rods were predominant in the gingival crevice (60.5%) and cocci in saliva (69.1%). Of the total organisms, nonsporing, gram-positive anaerobic rods accounted for 24% in the gingival crevice and 9.7% in saliva. These organisms were characterized on the basis of the type of fatty acids produced from glucose and various biochemical reactions. They belonged to the following genera: Actinomyces, Propionibacterium, Arachnia, Lactobacillus, Eubacterium, and Bifidobacterium. Bifidobacteria were present only in saliva. Although members of the other genera were present both in the gingival crevice and saliva, there were considerable differences in the proportion of any particular organism (in relation to the total anaerobic viable count) between the two sites. The result of this study also indicates a greater than previously appreciated level of Propionibacterium and Arachnia in the human mouth. PMID:646354

Sialic acids are monosaccharides typically found on cell surfaces and attached to soluble proteins, or as essential components of ganglioside structures that play a critical role in brain development and neural transmission. Human milk also contains sialic acid conjugated to oligosaccharides, glycolipids, and glycoproteins. These nutrients can reach the large bowel where they may be metabolised by the microbiota. However, little is known about the members of the microbiota involved in this function. To identify intestinal bacteria that utilise sialic acid within a complex intestinal community, we cultured the caecal microbiota from piglets in the presence of 13C-labelled sialic acid. Using RNA-based stable isotope probing, we identified bacteria that consumed 13C-sialic acid by fractionating total RNA in isopycnic buoyant density gradients followed by 16S rRNA gene analysis. Addition of sialic acid caused significant microbial community changes. A relative rise in Prevotella and Lactobacillus species was accompanied by a corresponding reduction in the genera Escherichia/Shigella, Ruminococcus and Eubacterium. Inspection of isotopically labelled RNA sequences suggests that the labelled sialic acid was consumed by a wide range of bacteria. However, species affiliated with the genus Prevotella were clearly identified as the most prolific users, as solely their RNA showed significantly higher relative shares among the most labelled RNA species. Given the relevance of sialic acid in nutrition, this study contributes to a better understanding of their microbial transformation in the intestinal tract with potential implications for human health. PMID:25816158

Limited knowledge of the structure and activities of the ruminal bacterial community prevents the understanding of the effect of population dynamics on functional bacterial groups and on host productivity. This study aimed to identify particular bacteria associated with host feed efficiency in steers with differing diets and residual feed intake (RFI) using culture-independent methods: PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis. PCR-DGGE profiles were generated from the ruminal fluid of 55 steers fed a low-energy-density diet and then switched to a high-energy-density diet. Bacterial profile comparisons by multivariate statistical analysis showed a trend only for RFI-related clusters on the high-energy diet. When steers (n = 19) belonging to the same RFI group under both diets were used to identify specific bacterial phylotypes related to feed efficiency traits, correlations were detected between dry matter intake, average daily gain, and copy numbers of the 16S rRNA gene of Succinivibrio sp. in low-RFI (efficient) steers, whereas correlations between Robinsoniella sp. and RFI (P < 0.05) were observed for high-RFI (inefficient) animals. Eubacterium sp. differed significantly (P < 0.05) between RFI groups that were only on the high-energy diet. Our work provides a comprehensive framework to understand how particular bacterial phylotypes contribute to differences in feed efficiency and ultimately influence host productivity, which may either depend on or be independent from diet factors. PMID:22156428